Method of using anti-CD47 antibody molecules

ABSTRACT

The invention relates to antibody molecules and antigen-binding portions thereof which bind specifically to CD47 (Cluster of Differentiation 47, also known as integrin associated protein [IAP]). In aspects of the invention, the anti-CD47 antibody molecules and antigen-binding portions thereof specifically bind to human CD47 and cynomolgus monkey CD47. Medical uses of the anti-CD47 antibody molecules and antigen-binding portions of the invention are disclosed. The anti-CD47 antibody molecules and antigen-binding portions of the invention represent modified and optimised binding molecules compared with a VxP037 murine/humanized anti-CD47 antibody described in WO2014/093678A2.

This application is a division of U.S. patent application Ser. No.16/543,884, filed on Aug. 19, 2019, and issued as U.S. Pat. No.10,683,350, which is a continuation of International Patent ApplicationNo. PCT/GB2018/052347, filed on Aug. 17, 2018, which claims the benefitof GB Patent Application No. 1808570.4, filed on May 24, 2018, GB PatentApplication No. 1802595.7, filed on Feb. 16, 2018 and GB PatentApplication No. 1713298.6, filed on Aug. 18, 2017, the disclosure ofeach of which is hereby incorporated by reference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:UH4L_001_02US_SeqListST25.txt, date recorded: May 19, 2020, file size˜80,851 bytes).

FIELD OF THE INVENTION

The invention relates to antibody molecules binding specifically to CD47(Cluster of Differentiation 47, also known as integrin associatedprotein [IAP]) and medical uses thereof.

BACKGROUND OF THE INVENTION

CD47 (also known as integrin associated protein [IAP]) is atransmembrane protein that belongs to the immunoglobulin superfamily andbinds to several known partners, including: membrane integrins,thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα).CD47 is associated with a range of cellular processes, includingapoptosis, proliferation, adhesion, and migration of cells and,importantly, it plays a key role in immune and angiogenic responses.CD47-SIRPα signalling is a critical molecular interaction that inhibitsthe activation of phagocytosis by macrophages and other myeloid cells.This promotes the survival of tumour cells and therefore acts as amyeloid lineage-specific immune checkpoint.

Preclinical evidence suggests that blocking CD47-SIRPα signalling canenhance the phagocytic activity of macrophages and inhibit the growth ofxenografts in numerous experimental models of both haematological andsolid malignancies. As macrophage activity is also a recognised factorin the biology of inflammation-associated tissue remodelling such astissue fibrosis and the formation of atherosclerotic plaques, theCD47-SIRPα signalling axis is also of considerable therapeutic potentialin non-cancerous diseases. Hence, anti-CD47 mAbs have the potential toact as immunotherapeutic agents in cancer and other settings, and toamplify the effectiveness of currently established therapies.

The majority of currently approved antibody therapeutics are derivedfrom immunized rodents. Many of those antibodies have undergone aprocess known as “humanization”, via the “grafting” of murine CDRs intohuman v-gene framework sequences (see Nelson et al., 2010, Nat Rev DrugDiscov 9: 767-774). This process is often inaccurate and leads to areduction in target binding affinity of the resulting antibody. Toreturn the binding affinity of the original antibody, murine residuesare usually introduced at key positions in the variable domainframeworks of the grafted v-domains (also known as “back-mutations”).

While antibodies humanized via CDR grafting and back mutations have beenshown to induce lower immune response rates in the clinic in comparisonto those with fully murine v-domains, antibodies humanized using thisbasic grafting method still carry significant clinical development risksdue to the potential physical instability and immunogenicity motifsstill housed in the grafted CDR loops. Antibodies such as CD47inhibitors that target receptors on immune cells, and whosepharmacological function is to stimulate immune responses via antigenpresentation, are at heightened risk of provoking anti-drug antibodyresponses. These anti-drug antibody responses in the patient can reducedrug half-life, potency and safety during clinical use. As animaltesting of protein immunogenicity is often non-predictive of immuneresponses in man, antibody engineering for therapeutic use focuses onminimizing predicted human T-cell epitope content, non-human germlineamino acid content and aggregation potential in the purified protein.

The ideal humanized antagonistic anti-CD47 antibody would therefore haveas many identical residues as possible in the v-domains to those foundin both the frameworks and CDRs of well-characterized human germlinesequences. Townsend et al. (2015; PNAS 112: 15354-15359) describe amethod for generating antibodies in which CDRs derived from rat, rabbitand mouse antibodies were grafted into preferred human frameworks andthen subject to a human germ-lining approach termed “Augmented BinarySubstitution”. Although the approach demonstrated a fundamentalplasticity in the original antibody paratopes, in the absence of highlyaccurate antibody-antigen co-crystal structural data, it is still notpossible to reliably predict which individual residues in the CDR loopsof any given antibody can be converted to human germline, and in whatcombination.

CDR germ-lining is thus a complex, multifactorial problem, as multiplefunctional properties of the molecule should preferably be maintained,including in this instance: target binding specificity, affinity to CD47from both human and animal test species (e.g. cynomolgus monkey, alsoknown as the crab-eating macaque, i.e. Macaca fascicularis), v-domainbiophysical stability and/or IgG expression yield. Antibody engineeringstudies have shown that mutation of even single residue positions in keyCDRs can have dramatic effects on all of these desired molecularproperties.

WO2014/093678A2 describes an antagonistic murine anti-CD47 IgG moleculetermed “VxP037”, and also the preparation of humanized forms of VxP037.Those humanized forms of VxP037 were produced using classicalhumanization techniques, i.e. by grafting of Kabat-defined murine CDRsinto human heavy and light chain framework sequences, with some of thehuman framework residues being potentially back-mutated to thecorrespondingly positioned VxP037 murine residues. For reasons notedabove, such humanized forms of VxP037 described in WO2014/093678A2 arenot ideal.

The present invention provides a number of optimized anti-CD47antibodies and medical uses thereof.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided an antibodymolecule which specifically binds to human CD47, and optionally also tocynomolgus monkey CD47 and/or to mouse CD47, or an antigen-bindingportion thereof, wherein the antibody molecule or antigen-bindingportion comprises a heavy chain variable region with:

an HCDR1 having amino acids in sequence in the following order: G-Y-T orany amino acid (for example, S, N or R)-F-T or a conservativesubstitution of T-N or a conservative substitution of N-Y-Y-I or aconservative substitution of I-F or any amino acid (for example, V or G)(SEQ ID NO: 1);an HCDR2 having amino acids in sequence in the following order: M or aconservative substitution of M-G-I or any amino acid (for example, N, Vor D)-I-N or any amino acid (for example, Y)-P-V or any amino acid (forexample, G or F)-D or a conservative substitution of D-G or aconservative substitution of G-D-T-N or a conservative substitution of N(for example, R)-Y or a conservative substitution of Y-N or aconservative substitution of N (for example, S)-P-S-F-Q-G (SEQ ID NO:2); andan HCDR3 having amino acids in sequence in the following order: G-G-Y orany amino acid (for example, H, I, Q or F)-T or any amino acid (forexample, V or I)-M or any amino acid (for example, T, R, P, A or L)-D orany amino acid (for example, G)-R or any amino acid (for example, Q, N,Y, S, W, K, A, E, F, H, I, L, M, T or V) (SEQ ID NO: 3).

In aspects of the invention, the HCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence GYTFTNYYVF (SEQ ID NO:4) (VxP037 murine/humanized antibody HCDR1 disclosed in WO2014/093678A2)and/or the HCDR3 of the antibody molecule or antigen-binding portion mayexclude the sequence GGYTMDY (SEQ ID NO: 5) (VxP037 murine/humanizedantibody HCDR3 disclosed in WO2014/093678A2).

The antibody molecule or antigen-binding portion may further comprise alight chain variable region with:

an LCDR1 having amino acids in sequence in the following order: R-S-S-Qor a conservative substitution of Q-S-L or a conservative substitutionof L-L or a conservative substitution of L-H-S-N or any amino acid (forexample, Q, S, T, A or G) or a conservative substitution of N (forexample, Q, S, T or G)-G or a conservative substitution of G (forexample, A)-Y or any amino acid (for example, N or S)-T or aconservative substitution of T (for example, N)-Y-L-H or any amino acid(for example, D) (SEQ ID NO: 6);an LCDR2 having amino acids in sequence in the following order: K or anyamino acid (for example, L or M)-V or any amino acid (for example,G)-S-N or any amino acid (for example, Y)-R-L or any amino acid (forexample, F, A or S)-S(SEQ ID NO: 7); and an LCDR3 having amino acids insequence in the following order: F or any amino acid (for example, L, M,S, T or V)-Q-Q or any amino acid (for example, N, A, T or S)-T or anyamino acid (for example, L, M or I)-H or a conservative substitution ofH-T or any amino acid (for example, V, I, A or F)-P or any amino acid(for example, L)-R or any amino acid (for example, W)-T (SEQ ID NO: 8).

In aspects of the invention, the LCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence RSSQSLVHSNGNTYLH (SEQID NO: 9) (VxP037 murine/humanized antibody LCDR1 disclosed inWO2014/093678A2), and/or the LCDR2 of the antibody molecule orantigen-binding portion may exclude the sequence KVSYRFS (SEQ ID NO: 10)(VxP037 murine/humanized antibody LCDR2 disclosed in WO2014/093678A2)and/or the LCDR3 of the antibody molecule or antigen-binding portion mayexclude the sequence SQNTHVPRT (SEQ ID NO: 11) (VxP037 murine/humanizedantibody LCDR3 disclosed in WO2014/093678A2).

The CDR sequences above are defined using the “Unified” definition, asset out in Table 1 and described below. As an alternative, the CDRsequences in the present invention may be defined using the shorter“AHo” definition (see Table 1), which is based on structural biology andaims to unify nomenclature for all immunoglobulin v-domains.

Using the shorter “AHo” CDR definition, the invention in one aspectprovides an antibody molecule which specifically binds to human CD47,and optionally also to cynomolgus monkey CD47 and/or to mouse CD47, oran antigen-binding portion thereof, wherein the antibody molecule orantigen-binding portion comprises a heavy chain variable region with: anHCDR1 having amino acids in sequence in the following order: G-S-G-Y-Tor any amino acid (for example, S, N or R)-F-T or a conservativesubstitution of T-N or a conservative substitution of N-Y-Y (SEQ ID NO:12);

an HCDR2 having amino acids in sequence in the following order: I-N orany amino acid (for example, Y)-P-V or any amino acid (for example, G orF)-D or a conservative substitution of D-G or a conservativesubstitution of G-D-T-N or a conservative substitution of N (forexample, R)-Y or a conservative substitution of Y-N or a conservativesubstitution of N (for example, S)-P-S-F-Q-G (SEQ ID NO: 13); andan HCDR3 having amino acids in sequence in the following order: G-G-Y orany amino acid (for example, H, I, Q or F)-T or any amino acid (forexample, V or I)-M or any amino acid (for example, T, R, P, A or L)-D orany amino acid (for example, G) (SEQ ID NO: 14).

Using the AHo definition, the HCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence GSGYTFTNYY (SEQ ID NO:15) (VxP037 murine/humanized antibody HCDR1 disclosed inWO2014/093678A2) and/or the HCDR3 of the antibody molecule orantigen-binding portion may exclude the sequence GGYTMD (SEQ ID NO: 16)(VxP037 murine/humanized antibody HCDR3 disclosed in WO2014/093678A2).

The antibody molecule or antigen-binding portion may further comprise alight chain variable region with CDRs defined using the AHo definitionas follows:

an LCDR1 having amino acids in sequence in the following order: S-S-Q ora conservative substitution of Q-S-L or a conservative substitution ofL-L or a conservative substitution of L-H-S-N or any amino acid (forexample, Q, S, T, A or G) or a conservative substitution of N (forexample, Q, S, T or G)-G or a conservative substitution of G (forexample, A)-Y or any amino acid (for example, N or S)-T or aconservative substitution of T (for example, N)-Y (SEQ ID NO: 17);an LCDR2 having amino acids in sequence in the following order: K or anyamino acid (for example, L or M)-V or any amino acid (for example,G)-S-N or any amino acid (for example, Y)-R-L or any amino acid (forexample, F, A or S)-S(SEQ ID NO: 7); andan LCDR3 having amino acids in sequence in the following order: Q or anyamino acid (for example, N, A, T or S)-T or any amino acid (for example,L, M or I)-H or a conservative substitution of H-T or any amino acid(for example, V, I, A or F)-P or any amino acid (for example, L)-R orany amino acid (for example, W) (SEQ ID NO: 18).

Using the AHo definition, in aspects of the invention the LCDR1 of theantibody molecule or antigen-binding portion may exclude the sequenceSSQSLVHSNGNTY (SEQ ID NO: 19) (VxP037 murine/humanized antibody LCDR1disclosed in WO2014/093678A2), and/or the LCDR2 of the antibody moleculeor antigen-binding portion may exclude the sequence KVSYRFS (SEQ ID NO:10) (VxP037 murine/humanized antibody LCDR2 disclosed inWO2014/093678A2) and/or the LCDR3 of the antibody molecule orantigen-binding portion may exclude the sequence NTHVPR (SEQ ID NO: 20)(VxP037 murine/humanized antibody LCDR3 disclosed in WO2014/093678A2).

Also provided according to the invention is an immunoconjugatecomprising the antibody molecule or antigen-binding portion thereof asdefined herein linked a therapeutic agent.

In another aspect the invention provides nucleic acid molecule encodingthe antibody molecule or antigen-binding portion thereof as definedherein.

Further provided is a vector comprising the nucleic acid molecule of theinvention.

Also provided is a host cell comprising the nucleic acid molecule or thevector of the invention as defined herein.

In a further aspect there is provided a method of producing an anti-CD47antibody and/or an antigen-binding portion thereof, comprising culturingthe host cell of the invention under conditions that result inexpression and/or production of the antibody and/or the antigen-bindingportion thereof, and isolating the antibody and/or the antigen-bindingportion thereof from the host cell or culture.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising the antibody molecule or antigen-binding portionthereof of the invention as defined herein, or the immunoconjugate ofthe invention as defined herein, or the nucleic acid molecule of theinvention as defined herein, or the vector of the invention as definedherein.

Further provided is a method for enhancing an immune response in asubject, comprising administering an effective amount of the antibodymolecule or antigen-binding portion thereof of the invention as definedherein, or the immunoconjugate of the invention as defined herein, orthe nucleic acid molecule of the invention as defined herein, or thevector of the invention as defined herein, or the pharmaceuticalcomposition of the invention as defined herein.

In a further aspect there is provided a method for treating orpreventing cancer in a subject, comprising administering an effectiveamount of the antibody molecule or antigen-binding portion thereof ofthe invention as defined herein, or the immunoconjugate of the inventionas defined herein, or the nucleic acid molecule of the invention asdefined herein, or the vector of the invention as defined herein, or thepharmaceutical composition of the invention as defined herein.

The invention also provides an antibody molecule or antigen-bindingportion thereof of the invention as defined herein, or theimmunoconjugate of the invention as defined herein, or the nucleic acidmolecule of the invention as defined herein, or the vector of theinvention as defined herein, or the pharmaceutical composition of theinvention as defined herein, for use in the treatment of cancer.

In another aspect the invention provides the antibody molecule, orantigen-binding portion thereof, or the immunoconjugate, or the nucleicacid molecule, or the vector for use, or the method of treatment of theinvention as defined herein, for separate, sequential or simultaneoususe in a combination combined with a second therapeutic agent, forexample an anti-cancer agent.

In a further aspect there is provided the use of an antibody molecule orantigen-binding portion thereof of the invention as defined herein, oran immunoconjugate of the invention as defined herein, or a nucleic acidmolecule of the invention as defined herein, or a vector of theinvention as defined herein, or a pharmaceutical composition of theinvention as defined herein, in the manufacture of a medicament for thetreatment of cancer.

The invention also provides a method for treating or preventing anischemia-reperfusion injury, an autoimmune disease or an inflammatorydisease in a subject, comprising administering an effective amount ofthe antibody molecule or antigen-binding portion thereof as definedherein, or the immunoconjugate as defined here, or the nucleic acidmolecule as defined herein, or the vector as defined herein, or thepharmaceutical composition as defined herein.

The autoimmune disease or inflammatory disease may be selected in allaspects from the group consisting of: arthritis, multiple sclerosis,psoriasis, Crohn's disease, inflammatory bowel disease, lupus, Grave'sdisease and Hashimoto's thyroiditis, and ankylosing spondylitis.

The ischemia-reperfusion injury in all aspect may occur in organtransplantation, acute kidney injury, cardiopulmonary bypass surgery,pulmonary hypertension, sickle cell disease, myocardial infarction,stroke, surgical resections and reconstructive surgery, reattachment ofan appendage or other body part, skin grafting or trauma.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of an ischemia-reperfusion injury, an autoimmune diseaseor an inflammatory disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of anischemia-reperfusion injury, an autoimmune disease or an inflammatorydisease.

The invention also provides a method for treating or preventing acardiovascular disease or a fibrotic disease in a subject, comprisingadministering an effective amount of the antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined here, or the nucleic acid molecule as definedherein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of a cardiovascular disease or a fibrotic disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of anischemia-reperfusion injury, an autoimmune disease, an inflammatorydisease or a fibrotic disease.

The cardiovascular disease in any aspect of the invention may forexample be coronary heart disease or atherosclerosis.

The fibrotic disease in any aspect of the invention may be selected fromthe group consisting of myocardial infarction, angina, osteoarthritis,pulmonary fibrosis, cystic fibrosis, bronchitis and asthma.

The invention also provides a method of producing an antibody moleculewhich specifically binds to human CD47 and optionally also to cynomolgusmonkey CD47 and/or to mouse CD47, or an antigen-binding portion thereof,comprising the steps of:

(1) grafting anti-CD47 CDRs from a non-human source into a humanv-domain framework to produce a humanized anti-CD47 antibody molecule orantigen-binding portion thereof;

(2) generating a phage library of clones of the humanized anti-CD47antibody molecule or antigen-binding portion thereof comprising one ormore mutations in the CDRs;

(3) screening the phage library for binding to human CD47 and optionallyalso to cynomolgus monkey CD47 and/or to mouse CD47;

(4) selecting clones from the screening step (3) having bindingspecificity to human CD47 and optionally also to cynomolgus monkey CD47and/or to mouse CD47; and

(5) producing an antibody molecule which specifically binds to humanCD47 and optionally also to cynomolgus monkey CD47 and/or to mouse CD47,or an antigen-binding portion thereof from clones selected from step(4).

The method may comprise a further step of producing additional clonesbased on the clones selected in step (4), for example based on furtherexploratory mutagenesis at specific positions in the CDRs of the clonesselected in step (4), to enhance humanization and/or minimise human Tcell epitope content and/or improve manufacturing properties in theantibody molecule or antigen-binding portion thereof produced in step(5).

The method may comprise a further step of assessing immunogenicity ofone or more v-domains in the clones selected in step (4) or the antibodymolecule produced in step (5), and optionally generating one or morefurther mutations, for example in a CDR and framework region, to reduceimmunogenicity. Immunogenicity may be assessed by identifying thelocation of T cell epitopes, for example using in silico technologies asdescribed herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-FIG. 1C. Direct binding ELISA of library-derived anti-CD47 scFvsagainst human and mouse CD47-Fc proteins. Clones were derived from 3separate phage selection branches (FIG. 1A shows Branch A periprepELISA; FIG. 1B shows Branch B periprep ELISA; and FIG. 1C shows Branch Cperiprep ELISA) where phage populations were selected on biotinylatedhuman, mouse and/or cynomolgus monkey CD47-Fc proteins in each round.After each round of selection, library-derived clones (black circles)were screened against both human and mouse CD47-Fc. Mean±SD values ineach round are represented in grey bars. In each graph, the X-axis showsselection round (“R”), with “H” denoting human and “M” denoting mouse,and the Y-axis shows binding signal (OD 450 nm).

FIG. 2A-FIG. 2B. Analysis of CDR residue tolerance for mutation togermline. A plot of murine amino acid retention frequencies in the CDRsof the ELISA-positive population of 854 unique scFv clones is shown forV_(H) (FIG. 2A) and V_(L) (FIG. 2B) domains, respectively. Only thoseresidues targeted for human/murine residue mutagenesis are plotted,other than in the HCDR3. In each plot, CDR residues are shown in theX-axis and the Y-axis shows percentage retention of each murine residue.CDR residues noted in parentheses on the X-axes were identical to thosefound in the human germlines used for grafting (IGKV2-28 and IGHV5-51).Those residues in the HCDR2 that are not in parentheses, but whosevalues are set at 0, were mutated to human germline during the graftingprocess. In both plots the dashed line in grey at 75% represents thecutoff for tolerance of murine residue replacement by human germline. InFIG. 2A, the sequences on the x-axis in order from left to right are SEQID NO: 4, SEQ ID NO: 302 and SEQ ID NO: 5. In FIG. 2B, the sequences onthe x-axis in order from left to right are SEQ ID NO: 9, SEQ ID NO: 10and SEQ ID NO: 11.

FIG. 3A-FIG. 3H. Direct titration ELISA for IgG binding to human, mouseand cyno CD47-Fc proteins. Chimeric anti-CD47 (mVH/mVL), library-derivedclones in human IgG1 null format were titrated (in μg/ml) in a directbinding ELISA against human, mouse and cyno CD47-Fc proteins (FIG.3A-FIG. 3H). The mVH/mVL, library-derived clones and designer clone MHdemonstrated binding activity against all 3 orthologs of CD47. CloneVH-A1/VL-B1 binds human and cyno CD47 but does not bind to mouse. CloneTTP lost almost all binding function. In each graph, the X-axis showsIgG concentration in μg/ml and the Y-axis shows binding signal (OD 450nm).

FIG. 4A-FIG. 4C. ELISA-based CD47-Fc-SIRPα competition assay. ELISAbinding signal for human (FIG. 4A), cyno (FIG. 4B) and mouse (FIG. 4C)CD47-Fc proteins to plate-bound human SIRPα was examined in the presenceof titrated competitor library-derived leads: A-D5, G-B6, D-H3 andVH-A1/VL-B1 in IgG1null format, Isotype IgG1 as a negative control, plusmVH/mVL in IgG1null format as a positive control. All library-derivedIgGs and mVH/mVL demonstrated concentration-dependent reduction inbinding of the human, murine and cyno CD47-Fc proteins, suggestingmaintenance of a shared epitope. Notably, clone A-D5 exhibitedsignificantly increased potency in neutralisation of mouse CD47 incomparison to the mVH/mVL and VH-A1/VL-B1 did not show the capacity toneutralise the activity of murine CD47. Neither designer clone MH norTTP exhibited any neutralisation signal and are not plotted here, forclarity. In each graph, the X-axis shows antibody concentration in nMand the Y-axis shows binding signal (OD 450 nm). In the figure legends,“IC” refers to isotype control.

FIG. 5A-FIG. 5G. Binding specificity analyses for prioritized leadclones. Off-target homologue binding risk for mVH/mVL in IgG1 (FIG. 5A)and IgG1null (FIG. 5B) format and library-derived leads A-D5 (FIG. 5C),VH-A1/VL-B1 (FIG. 5D), F-E7 (FIG. 5E), D-H3 (FIG. 5F), and G-B6 (FIG.5G) in IgG1null format was examined by direct ELISA on CD47-Fc orthologsand a panel of 14 human immunoglobulin superfamily proteins as labelledon each X-axis (“B” refers to blank). Binding to human, cyno and murineCD47-Fcs (h/c/mCD47-Fc) was performed at an IgG concentration of 1μg/ml. Binding to all other proteins was performed at an IgGconcentration of 10 μg/ml. In each plot, the Y-axis shows binding signal(OD 450 nm). For almost all IgGs, binding was observed to hCD47-Fc,mCD37-Fc and cCD47-Fc alone. No binding above background was observedfor any other human protein. Notably, clone VH-A1/VL-B1 again did notshow reactivity to murine CD47.

FIG. 6A-FIG. 6C. Flow cytometric binding to human and cyno CD47+ CHO-K1cells. Commercial anti-CD47 antibody MS1991, human IgG1 (“I IgG1”) andIgG4 (“I IgG4”) Isotype controls, lead library-derived IgGs in bothIgG1null (“IgG1N”) and IgG4(S228P) formats were examined for specificbinding on cyno-transfected CHO-K1 cells (FIG. 6A), human-transfectedCHO-K1 cells (FIG. 6B), and wild type (wt, i.e. untransfected) CHO-K1cells (FIG. 6C). IgGs were tested at concentrations ranging from24-100,000 ng/ml. Concentration-dependent binding was observed againstboth human and cyno cell lines for all CD47-specific antibodies but notIsotype controls. Low-level binding signals above background wereobserved against wild type CHO-K1 cells for most antibodies, withsignificantly stronger signal for the mVH/mVL-derived IgGs andparticularly low signal for VH-A1/VL-B1 IgGs. In each graph, the X-axisshows each IgG tested and its concentration in ng/ml, and the Y-axisshows mean fluorescence intensity (MFI).

FIG. 7. Flow cytometric testing of binding to human HL60 cells.Commercial anti-CD47 antibody MS1991, human IgG1 and IgG4 Isotypecontrols (“I IgG1” and “I IgG4”, respectively), lead library-derivedIgGs in both IgG1null (“IgG1N) and IgG4(S228P) formats were examined forspecific binding on HL60 cells. IgGs were tested at concentrationsranging from 24-100000 ng/ml. Concentration-dependent binding wasobserved for all clones other than the Isotype controls. In each graph,the X-axis shows each IgG tested and its concentration in ng/ml, and theY-axis shows mean fluorescence intensity (MFI).

FIG. 8A-FIG. 8C. Development risk ELISAs. This assay showed that theA-D5, G-B6, D-H3, VH-A1/VL-B1 and mVH/mVL antibodies in IgG1null formexhibit little or no binding to the negatively charged biomoleculesInsulin (FIG. 8A), double-stranded DNA (dsDNA) (FIG. 8B) andsingle-stranded DNA (ssDNA) (FIG. 8C). In each graph, the X-axis showsIgG concentration in μg/ml and the Y-axis shows binding signal (OD 450nm). Strong off-target binding to these molecules, as observed forBococizumab and Briakinumab analogues has been shown to be a high-riskindicator of poor clinical performance of therapeutic antibodies.

FIG. 9A-FIG. 9C. Direct titration ELISA for designer IgGs binding tohuman, mouse and cyno CD47-Fc proteins. Chimeric anti-CD47 (mVH/mVL),designer A-D5-derived clones in human IgG1null format were titrated (inμg/ml) in a direct binding ELISA against human (FIG. 9A), cyno (FIG. 9B)and mouse (FIG. 9C) CD47-Fc proteins. In each graph, the X-axis showsIgG concentration in μg/ml and the Y-axis shows binding signal (OD 450nm). Most clones demonstrated binding activity against all 3 orthologsof CD47, although clone A-D5.7 only shows weak binding to cyno CD47 andclone A-D5.10 lost almost all binding function to mouse CD47. In thefigure legend, “IgG1NI” refers to IgG1 null isotype.

FIG. 10A-FIG. 10C. ELISA-based CD47-Fc-SIRPα competition assay fordesigner IgGs. ELISA binding signal for human (FIG. 10A), cyno (FIG.10B) and mouse (FIG. 10C) CD47-Fc proteins to plate-bound human SIRPαwas examined in the presence of titrated competitor designer IgGs inIgG1null format, plus Isotype IgG1 as a negative control (represented by“IgG1NI”) and mVH/mVL in IgG1null format as a positive control. In eachgraph, the X-axis shows IgG concentration in nM and the Y-axis showsbinding signal (OD 450 nm). Notably, several A-D5-derived clones againexhibited significantly increased potency in neutralisation of mouseCD47 in comparison to the mVH/mVL. Neither designer clone A-D5.7 norA-D5.10 exhibited any neutralisation signal on the orthologs for whichthey showed weak ELISA binding signal and are not plotted here, forclarity.

FIG. 11A-FIG. 11C. Direct titration ELISA for A-D5.4-derived designerIgGs binding to human, mouse and cyno CD47-Fc proteins. Chimericanti-CD47 (mVH/mVL), designer A-D5.4-derived clones in human IgG1 nullformat were titrated (in μg/ml) in a direct binding ELISA against human(FIG. 11A), cyno (FIG. 11B) and mouse (FIG. 11C) CD47-Fc proteins. Ineach graph, the X-axis shows IgG concentration in μg/ml and the Y-axisshows binding signal (OD 450 nm). All clones demonstrated bindingactivity against all 3 orthologs of CD47. In the figure legends,“IgG1NI” refers to IgG1 null isotype.

FIG. 12A-FIG. 12C. ELISA-based CD47-Fc-SIRPα competition assay fordesigner IgGs. ELISA binding signal for human (FIG. 12A), cyno (FIG.12B) and mouse (FIG. 12C) CD47-Fc proteins to plate-bound human SIRPαwas examined in the presence of titrated competitor designer IgGs inIgG1null format, plus Isotype IgG1 as a negative control (represented by“IgG1NI”) and mVH/mVL in IgG1null format as a positive control. In eachgraph, the X-axis shown IgG concentration in nM and the Y-axis showsbinding signal (OD 450 nm). Notably, several A-D5.4-derived clones againexhibited significantly increased potency in neutralisation of mouseCD47 in comparison to the mVH/mVL.

FIG. 13A-FIG. 13B. Binding specificity analyses for designer clonesA-D5.4 and A-D5.16. Off-target homologue binding risk for A-D5.4 (FIG.13A) and A-D5.16 (FIG. 13B) in IgG1null format was examined by directELISA on CD47-Fc orthologs and a panel of 14 human immunoglobulinsuperfamily proteins (as labelled on each X-axis; “B” refers to blank).Binding to all proteins was performed at an IgG concentration of 10μg/ml. In each plot, the Y-axis shows binding signal (OD 450 nm). Forboth IgGs, binding was observed to hCD47-Fc, mCD37-Fc and cCD47-Fcalone. No binding above background was observed for any other humanprotein.

FIG. 14A-FIG. 14C. Development risk ELISAs for designer clones A-D5.4and A-D5.16. This assay showed that the A-D5.4 and A-D5.16 antibodies inIgG1 null form exhibit low (below negative control, Ustekinumab) bindingto the negatively charged biomolecules Insulin (FIG. 14A),double-stranded DNA (dsDNA) (FIG. 14B) and single-stranded DNA (ssDNA)(FIG. 14C). In each graph, the X-axis shows IgG concentration in μg/mland the Y-axis shows binding signal (OD 450 nm). Strong off-targetbinding to these molecules, as observed for Bococizumab and Briakinumabanalogues has been shown to be a high-risk indicator of poor clinicalperformance of therapeutic antibodies.

FIG. 15. Flow cytometric binding of library-derived and designer IgGs toCHO-K1 cells. Commercial anti-CD47 antibody MS1991, human IgG1 and IgG4Isotype controls (represented by “I IgG1” and “I IgG4” respectively),and lead IgGs A-D5, A-D5.4 and A-D5.16 in both IgG1null and IgG4 formatswere examined for specific binding on wild type (i.e. untransfected)CHO-K1 cells. IgGs were tested at concentrations ranging from 24-25,000ng/ml. Concentration-dependent binding was observed for the parentalmVH/mVL antibody in both IgG1null and IgG4 formats, but only weak or nobinding was observed for Isotype controls, MS1991 and IgGs A-D5, A-D5.4and A-D5.16 in both IgG formats. In each graph, the X-axis showsconcentration of IgG in ng/ml, and the Y-axis shows MFI.

FIG. 16. Flow cytometric testing of binding to human HL60 cells.Commercial anti-CD47 antibody MS1991, human IgG1 and IgG4 Isotypecontrols (represented by “I IgG1” and “I IgG4”, respectively), lead IgGsin both IgG1null and IgG4(S228P) formats were examined for specificbinding on HL60 cells. IgGs were tested at concentrations ranging from24-100000 ng/ml. Concentration-dependent binding was observed for allclones other than the Isotype controls. In each graph, the X-axis showsconcentration of IgG in ng/ml, and the Y-axis shows MFI.

FIG. 17. T cell epitope peptide content in lead antibody v-domains. Thev-domains of mVH/mVL, A-D5, A-D5.4 A-D5.16 and A-D5.16-DI antibodieswere examined for the presence of Germline (GE), High Affinity Foreign(HAF), Low Affinity Foreign (LAF) and TCED+ T cell receptor epitopes.Both the VH and VL domains of mVH/mVL were found to contain multiplehigh risk human T cell epitopes and few germline epitopes. In all leadclones, the high risk epitope content was significantly reduced andgermline epitope content significantly improved.

FIG. 18A-FIG. 18C. Direct titration ELISA for A-D5.16 and A-D5.16-DIdesigner IgGs binding to human, mouse and cyno CD47-Fc proteins.Chimeric anti-CD47 (mVH/mVL), designer A-D5.16 and A-D5.16-DI clones inhuman IgG1null format were titrated (in μg/ml) in a direct binding ELISAagainst human (FIG. 18A), cyno (FIG. 18B) and mouse (FIG. 18C) CD47-Fcproteins. All clones demonstrated binding activity against all 3orthologs of CD47. In each graph, the X-axis shows concentration of IgGin μg/ml, and the Y-axis shows binding signal (OD 450 nm).

FIG. 19A-FIG. 19C. ELISA-based CD47-Fc-SIRPα competition assay fordesigner IgGs. ELISA binding signal for human (FIG. 19A), cyno (FIG.19B) and mouse (FIG. 19C) CD47-Fc proteins to plate-bound human SIRPαwas examined in the presence of titrated competitor designer IgGs inIgG1null format, plus Isotype IgG1 as a negative control and mVH/mVL inIgG1 null format as a positive control. In each graph, the X-axis showsantibody concentration in nM, and the Y-axis shows binding signal (OD450 nm).

FIG. 20A-FIG. 20B. Flow cytometry phagocytosis analyses. (FIG. 20A) Flowcytometric analysis of the phagocytosis of CSFE-labelled HL60 cells byhuman CD14+ macrophages was performed at multiple concentrations (asshown on the X-axis) for clones A-D5, A-D5.4. A-D5.16 and mVH/mVL inIgG4 (S228P) format and additionally A-D5 in IgG1null format(represented by “IgG1 A-D5N”). The X-axis shows antibody concentration(μg/ml), and the Y-axis shows % cells that are CFSE⁺ and CD14⁺. (FIG.20B) The analysis was then repeated across multiple human macrophagedonors for A-D5 and mVH/mVL in IgG4 format, at a standard concentrationof 10 μg/ml. The X-axis shows donor number, and the Y-axis shows % cellsthat are CFSE⁺ and CD14⁺. “V” denotes vehicle.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention, there is provided anantibody molecule which specifically binds to human CD47 and optionallyalso to cynomolgus monkey CD47 and/or to mouse CD47, or anantigen-binding portion thereof, wherein the antibody molecule orantigen-binding portion comprises a heavy chain variable region with:

an HCDR1 having amino acids in sequence in the following order: G-Y-T orany amino acid (for example, S, N or R)-F-T or a conservativesubstitution of T-N or a conservative substitution of N-Y-Y-I or aconservative substitution of I-F or any amino acid (for example, V or G)(SEQ ID NO: 1);an HCDR2 having amino acids in sequence in the following order: M or aconservative substitution of M-G-I or any amino acid (for example, N, Vor D)-I-N or any amino acid (for example, Y)-P-V or any amino acid (forexample, G or F)-D or a conservative substitution of D-G or aconservative substitution of G-D-T-N or a conservative substitution of N(for example, R)-Y or a conservative substitution of Y-N or aconservative substitution of N (for example, S)-P-S-F-Q-G (SEQ ID NO:2); andan HCDR3 having amino acids in sequence in the following order: G-G-Y orany amino acid (for example, H, I, Q or F)-T or any amino acid (forexample, V or I)-M or any amino acid (for example, T, R, P, A or L)-D orany amino acid (for example, G)-R or any amino acid (for example, Q, N,Y, S, W, K, A, E, F, H, I, L, M, T or V) (SEQ ID NO: 3).

In aspects of the invention, the HCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence GYTFTNYYVF (SEQ ID NO:4) (VxP037 murine/humanized antibody HCDR1 disclosed in WO2014/093678A2)and/or the HCDR3 of the antibody molecule or antigen-binding portion mayexclude the sequence GGYTMDY (SEQ ID NO: 5) (VxP037 murine/humanizedantibody HCDR3 disclosed in WO2014/093678A2).

The antibody molecule or antigen-binding portion thereof according tothe invention may further comprise a light chain variable region with:

an LCDR1 having amino acids in sequence in the following order: R-S-S-Qor a conservative substitution of Q-S-L or a conservative substitutionof L-L or a conservative substitution of L-H-S-N or any amino acid (forexample, Q, S, T, A or G) or a conservative substitution of N (forexample, Q, S, T or G)-G or a conservative substitution of G (forexample, A)-Y or any amino acid (for example, N or S)-T or aconservative substitution of T (for example, N)-Y-L-H or any amino acid(for example, D) (SEQ ID NO: 6);an LCDR2 having amino acids in sequence in the following order: K or anyamino acid (for example, L or M)-V or any amino acid (for example,G)-S-N or any amino acid (for example, Y)-R-L or any amino acid (forexample, F, A or S)-S(SEQ ID NO: 7); andan LCDR3 having amino acids in sequence in the following order: F or anyamino acid (for example, L, M, S, T or V)-Q-Q or any amino acid (forexample, N, A, T or S)-T or any amino acid (for example, L, M or I)-H ora conservative substitution of H-T or any amino acid (for example, V, I,A or F)-P or any amino acid (for example, L)-R or any amino acid (forexample, W)-T (SEQ ID NO: 8).

In aspects of the invention, the LCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence RSSQSLVHSNGNTYLH (SEQID NO: 9) (VxP037 murine/humanized antibody LCDR1 disclosed inWO2014/093678A2), and/or the LCDR2 of the antibody molecule orantigen-binding portion may exclude the sequence KVSYRFS (SEQ ID NO: 10)(VxP037 murine/humanized antibody LCDR2 disclosed in WO2014/093678A2)and/or the LCDR3 of the antibody molecule or antigen-binding portion mayexclude the sequence SQNTHVPRT (SEQ ID NO: 11) (VxP037 murine/humanizedantibody LCDR3 disclosed in WO2014/093678A2).

The CDR sequences above are defined using the “Unified” definition, asset out in Table 1. As an alternative, the CDR sequences in the presentinvention may be defined using the shorter “AHo” definition (see Table1), which is based on structural biology and aims to unify nomenclaturefor all immunoglobulin v-domains.

Using the shorter “AHo” definition, the invention in one aspect providesan antibody molecule which specifically binds to human CD47, andoptionally also to cynomolgus monkey CD47 and/or to mouse CD47, or anantigen-binding portion thereof, wherein the antibody molecule orantigen-binding portion comprises a heavy chain variable region with:

an HCDR1 having amino acids in sequence in the following order:G-S-G-Y-T or any amino acid (for example, S, N or R)-F-T or aconservative substitution of T-N or a conservative substitution of N-Y-Y(SEQ ID NO: 12);

an HCDR2 having amino acids in sequence in the following order: I-N orany amino acid (for example, Y)-P-V or any amino acid (for example, G orF)-D or a conservative substitution of D-G or a conservativesubstitution of G-D-T-N or a conservative substitution of N (forexample, R)-Y or a conservative substitution of Y-N or a conservativesubstitution of N (for example, S)-P-S-F-Q-G (SEQ ID NO: 13); andan HCDR3 having amino acids in sequence in the following order: G-G-Y orany amino acid (for example, H, I, Q or F)-T or any amino acid (forexample, V or I)-M or any amino acid (for example, T, R, P, A or L)-D orany amino acid (for example, G) (SEQ ID NO: 14).

Using the AHo definition, the HCDR1 of the antibody molecule orantigen-binding portion may exclude the sequence GSGYTFTNYY (SEQ ID NO:15) (VxP037 murine/humanized antibody HCDR1 disclosed inWO2014/093678A2) and/or the HCDR3 of the antibody molecule orantigen-binding portion may exclude the sequence GGYTMD (SEQ ID NO: 16)(VxP037 murine/humanized antibody HCDR3 disclosed in WO2014/093678A2).

The antibody molecule or antigen-binding portion may further comprise alight chain variable region with:

an LCDR1 having amino acids in sequence in the following order: S-S-Q ora conservative substitution of Q-S-L or a conservative substitution ofL-L or a conservative substitution of L-H-S-N or any amino acid (forexample, Q, S, T, A or G) or a conservative substitution of N (forexample, Q, S, T or G)-G or a conservative substitution of G (forexample, A)-Y or any amino acid (for example, N or S)-T or aconservative substitution of T (for example, N)-Y (SEQ ID NO: 17);an LCDR2 having amino acids in sequence in the following order: K or anyamino acid (for example, L or M)-V or any amino acid (for example,G)-S-N or any amino acid (for example, Y)-R-L or any amino acid (forexample, F, A or S)-S(SEQ ID NO: 7); andan LCDR3 having amino acids in sequence in the following order: Q or anyamino acid (for example, N, A, T or S)-T or any amino acid (for example,L, M or 1)-H or a conservative substitution of H-T or any amino acid(for example, V, I, A or F)-P or any amino acid (for example, L)-R orany amino acid (for example, W) (SEQ ID NO: 18).

Using the AHo definition, in aspects of the invention the LCDR1 of theantibody molecule or antigen-binding portion may exclude the sequenceSSQSLVHSNGNTY (SEQ ID NO: 19) (VxP037 murine/humanized antibody LCDR1disclosed in WO2014/093678A2), and/or the LCDR2 of the antibody moleculeor antigen-binding portion may exclude the sequence KVSYRFS (SEQ ID NO:10) (VxP037 murine/humanized antibody LCDR2 disclosed inWO2014/093678A2) and/or the LCDR3 of the antibody molecule orantigen-binding portion may exclude the sequence NTHVPR (SEQ ID NO: 20)(VxP037 murine/humanized antibody LCDR3 disclosed in WO2014/093678A2).

As elaborated herein, the present inventors have succeeded for the firsttime in generating a number of optimized anti-CD47 antibody moleculesusing CDR sequences derived from the murine anti-CD47 antibody VxP037disclosed in WO2014/093678A2. In embodiments of the present invention,these antibody molecules have been selected to have binding specificityto both human CD47 as well as cynomolgus monkey CD47 and, for someclones, also to mouse CD47 (to facilitate studies in an animal testspecies). Further refining of the optimized antibody molecules asdescribed herein has provided improved binding to the mouse orthologueof CD47, improved potency in neutralisation of mouse CD47-SIRPαsignalling, improved variable domain stability, high expression yields,and/or reduced immunogenicity. For example, we demonstrate herein thatthe progenitor molecule for the murine anti-CD47 antibody VxP037 carriestwo major immunogenicity risks in the LCDR1 and LCDR2 that will becarried through with classical humanization techniques (as used inWO2014/093678A2) but are ameliorated in optimized antibody molecules asdescribed herein.

The antibody molecule or antigen-binding portion of the presentinvention may have improved in silico immunogenicity compared with anantibody molecule comprising the CDR sequences of SEQ ID NOs: 4 (HCDR1),123 (HCDR2), 5 (HCDR3), 9 (LCDR1), 10 (LCDR2) and 11 (LCDR3).

The antibody molecule or antigen-binding portion of the presentinvention may exhibit no binding to hamster CD47, or exhibit reducedbinding to hamster CD47 compared with an antibody molecule comprisingthe CDR sequences of SEQ ID NOs: 4 (HCDR1), 123 (HCDR2), 5 (HCDR3), 9(LCDR1), 10 (LCDR2) and 11 (LCDR3). For example, the antibody moleculeor antigen-binding portion of the present invention may exhibit nobinding to CHO cells as measured by flow cytometry, or reduced bindingto CHO cells as measured by flow cytometry, compared with an antibodycomprising the CDR sequences of SEQ ID NOs: 4 (HCDR1), 123 (HCDR2), 5(HCDR3), 9 (LCDR1), 10 (LCDR2) and 11 (LCDR3). As shown in FIG. 15,representative antibody molecules of the present invention have littleor no cross-reactivity with CHO cells, whereas the original murinev-domains of clone mVH/mVL in either IgG1null or IgG4 format drivestrong, concentration-dependent binding to CHO cells.

Preferred optimized anti-CD47 antibody molecules of the presentinvention do not necessarily have the maximum number of human germlinesubstitutions at corresponding murine CDR or other (such as framework)amino acid positions. As elaborated in the experimental section below,we have found that “maximally humanized” antibody molecules are notnecessary “maximally optimized” in terms of anti-CD47 bindingcharacteristics and/or other desirable features.

The present invention encompasses modifications to the amino acidsequence of the antibody molecule or antigen-binding portion thereof asdefined herein. For example, the invention includes antibody moleculesand corresponding antigen-binding portions thereof comprisingfunctionally equivalent variable regions and CDRs which do notsignificantly affect their properties as well as variants which haveenhanced or decreased activity and/or affinity. For example, the aminoacid sequence may be mutated to obtain an antibody with the desiredbinding affinity to CD47. Insertions which include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues, areenvisaged. Examples of terminal insertions include an antibody moleculewith an N-terminal methionyl residue or the antibody molecule fused toan epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

The antibody molecule or antigen-binding portion of the invention mayinclude glycosylated and nonglycosylated polypeptides, as well aspolypeptides with other post-translational modifications, such as, forexample, glycosylation with different sugars, acetylation, andphosphorylation. The antibody molecule or antigen-binding portion of theinvention may be mutated to alter such post-translational modifications,for example by adding, removing or replacing one or more amino acidresidues to form or remove a glycosylation site.

The antibody molecule or antigen-binding portion of the invention may bemodified for example by amino acid substitution to remove potentialproteolytic sites in the antibody.

In the antibody molecule or antigen-binding portion thereof, the HCDR1may have the amino acid sequence: G-Y-T/S/N/R-F-T/N-N/S-Y-Y-I/V-F/V/G(SEQ ID NO: 21); the HCDR2 may have the amino acid sequence:M/I-G-V/N/I/D-I-N/Y-P-V/G/F-N/D-G/S-D-T-N/R/K-F/Y-N/S-P-S-F-Q-G (SEQ IDNO: 22); and the HCDR3 may have the amino acid sequence:G-G-F/H/I/Q/Y-T/VII-M/T/R/P/A/L-DIG-Y/Q/N/R/S/W/K/A/E/F/H/I/L/M/T/V (SEQID NO: 23). Alternatively, using the AHo definition, in the antibodymolecule or antigen-binding portion thereof, the HCDR1 may have theamino acid sequence: G-S-G-Y-T/S/N/R-F-T/N-N/S-Y-Y (SEQ ID NO: 24); theHCDR2 may have the amino acid sequence:I-N/Y-P-V/G/F-N/D-G/S-D-T-N/R/K-F/Y-N/S-P-S-F-Q-G (SEQ ID NO: 25); andthe HCDR3 may have the amino acid sequence:G-G-F/H/I/Q/Y-T/VII-M/T/R/P/A/L-DIG (SEQ ID NO: 26).

For example, the HCDR1 may have the amino acid sequence:G-Y-T/S-F-T-N-Y-Y-I-F (SEQ ID NO: 27); the HCDR2 may have the amino acidsequence: M/I-G-I/D-I-N-P-V-N/D-G-D-T-N/R-F/Y-N/S-P-S-F-Q-G (SEQ ID NO:28); and the HCDR3 may have the amino acid sequence:G-G-F/Y-T-M/P-D-Y/R/K/I (SEQ ID NO: 29). Alternatively, using the AHodefinition, the HCDR1 may have the amino acid sequence:G-S-G-Y-T/S-F-T-N-Y-Y (SEQ ID NO: 30); the HCDR2 may have the amino acidsequence: I-N-P-V-N/D-G-D-T-N/R-F/Y-N/S-P-S-F-Q-G (SEQ ID NO: 31); andthe HCDR3 may have the amino acid sequence: G-G-F/Y-T-M/P-D (SEQ ID NO:32).

In the antibody molecule or antigen-binding portion thereof, the LCDR1may have the amino acid sequence:R-S-S-Q/H-S-F/L-L/V-H-S-N/Q/A-G/A-Y/N/S-N/T-Y-L-H/D (SEQ ID NO: 33); theLCDR2 may have the amino acid sequence: L/K/M-V/G-S-N/Y-R-A/F/L/S-S(SEQID NO: 34); and the LCDR3 may have the amino acid sequence:F/L/M/S/T/V-Q-Q/N/A/T/S-T/L/M/I-Q/H-T/V/I/A/F-P/L-R/W-T (SEQ ID NO: 35).Alternatively, using the AHo definition, in the antibody molecule orantigen-binding portion thereof, the LCDR1 may have the amino acidsequence: S-S-Q/H-S-F/L-L/V-H-S-N/Q/A-G/A-Y/N/S-N/T-Y (SEQ ID NO: 36);the LCDR2 may have the amino acid sequence:L/K/M-V/G-S-N/Y-R-A/F/L/S-S(SEQ ID NO: 34); and the LCDR3 may have theamino acid sequence: Q/N/A/T/S-T/L/M/I-Q/H-T/V/I/A/F-P/L-R/W (SEQ ID NO:37).

For example, the LCDR1 may have the amino acid sequence:R-S-S-Q-S-L-L/V-H-S-N/Q/A-G-Y/N-N/T-Y-L-H/D (SEQ ID NO: 38); the LCDR2may have the amino acid sequence: L/K-V/G-S-N/Y-R-A/F/L-S(SEQ ID NO:39); and the LCDR3 may have the amino acid sequence:F/S-Q-Q/N/A-T/L-Q/H-T/V-P-R-T (SEQ ID NO: 40). Alternatively, using theAHo definition, the LCDR1 may have the amino acid sequence:S-S-Q-S-L-L/V-H-S-N/Q/A-G-Y/N-N/T-Y (SEQ ID NO: 41); the LCDR2 may havethe amino acid sequence: L/K-V/G-S-N/Y-R-A/F/L-S(SEQ ID NO: 39); and theLCDR3 may have the amino acid sequence: Q/N/A-T/L-Q/H-T/V-P-R (SEQ IDNO: 42).

In specific embodiments of the invention as defined using the UnifiedCDR definition, the antibody molecule or antigen-binding portion maycomprise:

(a) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGDINPVNGDTNYSPSFQG (SEQ ID NO: 44) (HCDR2), GGYTPDY (SEQ ID NO: 45)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KGSNRFS (SEQ ID NO:47) (LCDR2) and SQNLHVPRT (SEQ ID NO: 48) (LCDR3) [Clone D-H3]; or(b) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5]; or(c) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),IGDINPVNGDTNFSPSFQG (SEQ ID NO: 55) (HCDR2), GGYTMDK (SEQ ID NO: 56)(HCDR3), RSSQSLVHSNGYTYLH (SEQ ID NO: 57) (LCDR1), KGSYRAS (SEQ ID NO:58) (LCDR2) and SQNTQTPRT (SEQ ID NO: 59) (LCDR3) [Clone G-B6]; or(d) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVNGDTNYNPSFQG (SEQ ID NO: 60) (HCDR2), GGYTMGK (SEQ ID NO: 61)(HCDR3), RSSQSLVHSNGNTYLD (SEQ ID NO: 62) (LCDR1), KGSYRFS (SEQ ID NO:63) (LCDR2) and SQATHTPRT (SEQ ID NO: 64) (LCDR3) [Clone F-E7]; or(e) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGFTMDY (SEQ ID NO: 66)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KGSNRAS (SEQ ID NO:67) (LCDR2) and SQNTHTPRT (SEQ ID NO: 68) (LCDR3) [Clone VH-A1/VL-B1];or(f) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),IGIINPVDGDTRYSPSFQG (SEQ ID NO: 69) (HCDR2), GGYTMDI (SEQ ID NO: 70)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), LGSNRFS (SEQ ID NO:71) (LCDR2) and SQNTQTPRT (SEQ ID NO: 59) (LCDR3) [Clone MH]; or(g) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDI (SEQ ID NO: 70)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), LGSNRAS (SEQ ID NO:72) (LCDR2) and SQATQTPRT (SEQ ID NO: 73) (LCDR3) [Clone TTP]; or(h) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.1]; or(i) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYNPSFQG (SEQ ID NO: 74) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.2]; or(j) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.3]; or(k) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.4]; or(l) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KGSNRLS (SEQ ID NO:75) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.5]; or(m) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KGSNRLS (SEQ ID NO:75) (LCDR2) and FQNTQTPRT (SEQ ID NO: 76) (LCDR3) [Clone A-D5.6]; or(n) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), LGSNRLS (SEQ ID NO:77) (LCDR2) and FQNTQTPRT (SEQ ID NO: 76) (LCDR3) [Clone A-D5.7]; or(o) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSQGYTYLH (SEQ ID NO: 78) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.8]; or(p) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQQTHTPRT (SEQ ID NO: 79) (LCDR3) [Clone A-D5.9]; or(q) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSQGYTYLH (SEQ ID NO: 78) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQQTHTPRT (SEQ ID NO: 79) (LCDR3) [Clone A-D5.10]; or(r) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.11]; or(s) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYNPSFQG (SEQ ID NO: 74) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.12]; or(t) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYNYLH (SEQ ID NO: 46) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.13]; or(u) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSSGYNYLH (SEQ ID NO: 80) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.14]; or(v) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSGGYNYLH (SEQ ID NO: 81) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.15]; or(w) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSAGYNYLH (SEQ ID NO: 82) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.16]; or(x) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSTGYNYLH (SEQ ID NO: 83) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.17]; or(y) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNAYNYLH (SEQ ID NO: 84) (LCDR1), KVSNRLS (SEQ ID NO:53) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.18]; or(z) the amino acid sequences GYSFTNYYIF (SEQ ID NO: 43) (HCDR1),MGIINPVDGDTRYSPSFQG (SEQ ID NO: 65) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSAGYNYLH (SEQ ID NO: 82) (LCDR1), KVSNRFS (SEQ ID NO:85) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5.16-DI]; or(z.1) the amino acid sequences GYTFTNYYIF (SEQ ID NO: 49) (HCDR1),MGIINPVDGDTNYNPSFQG (SEQ ID NO: 50) (HCDR2), GGYTMDR (SEQ ID NO: 51)(HCDR3), RSSQSLLHSNGYTYLH (SEQ ID NO: 52) (LCDR1), KVSNRFS (SEQ ID NO:85) (LCDR2) and FQNTHTPRT (SEQ ID NO: 54) (LCDR3) [Clone A-D5-DI].

In the above specific embodiments of the invention, the CDRs mayalternatively be defined using the AHo CDR definition such that theantibody molecule or antigen-binding portion comprises:

(a) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVNGDTNYSPSFQG (SEQ ID NO: 87) (HCDR2), GGYTPD (SEQ ID NO: 88)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRFS (SEQ ID NO: 47)(LCDR2) and NLHVPR (SEQ ID NO: 90) (LCDR3) [Clone D-H3]; or(b) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5]; or(c) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVNGDTNFSPSFQG (SEQ ID NO: 94) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLVHSNGYTY (SEQ ID NO: 95) (LCDR1), KGSYRAS (SEQ ID NO: 58)(LCDR2) and NTQTPR (SEQ ID NO: 96) (LCDR3) [Clone G-B6]; or(d) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVNGDTNYNPSFQG (SEQ ID NO: 97) (HCDR2), GGYTMG (SEQ ID NO: 98)(HCDR3), SSQSLVHSNGNTY (SEQ ID NO: 19) (LCDR1), KGSYRFS (SEQ ID NO: 63)(LCDR2) and ATHTPR (SEQ ID NO: 99) (LCDR3) [Clone F-E7]; or(e) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGFTMD (SEQ ID NO: 101)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRAS (SEQ ID NO: 67)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone VH-A1/VL-B1]; or(f) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), LGSNRFS (SEQ ID NO: 71)(LCDR2) and NTQTPR (SEQ ID NO: 96) (LCDR3) [Clone MH]; or(g) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), LGSNRAS (SEQ ID NO: 72)(LCDR2) and ATQTPR (SEQ ID NO: 102) (LCDR3) [Clone TTP]; or(h) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.1]; or(i) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYNPSFQG (SEQ ID NO: 103) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.2]; or(j) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.3]; or(k) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.4]; or(l) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRLS (SEQ ID NO: 75)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.5]; or(m) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KGSNRLS (SEQ ID NO: 75)(LCDR2) and NTQTPR (SEQ ID NO: 96) (LCDR3) [Clone A-D5.6]; or(n) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), LGSNRLS (SEQ ID NO: 77)(LCDR2) and NTQTPR (SEQ ID NO: 96) (LCDR3) [Clone A-D5.7]; or(o) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.8]; or(p) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and QTHTPR (SEQ ID NO: 105) (LCDR3) [Clone A-D5.9]; or(q) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSQGYTY (SEQ ID NO: 104) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and QTHTPR (SEQ ID NO: 105) (LCDR3) [Clone A-D5.10]; or(r) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.11]; or(s) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYNPSFQG (SEQ ID NO: 103) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.12]; or(t) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYNY (SEQ ID NO: 89) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.13]; or(u) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSSGYNY (SEQ ID NO: 106) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.14]; or(v) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSGGYNY (SEQ ID NO: 107) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.15]; or(w) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.16]; or(x) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSTGYNY (SEQ ID NO: 109) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.17]; or(y) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNAYNY (SEQ ID NO: 110) (LCDR1), KVSNRLS (SEQ ID NO: 53)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.18]; or(z) the amino acid sequences GSGYSFTNYY (SEQ ID NO: 86) (HCDR1),INPVDGDTRYSPSFQG (SEQ ID NO: 100) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSAGYNY (SEQ ID NO: 108) (LCDR1), KVSNRFS (SEQ ID NO: 85)(LCDR2) and NTHTPR (SEQ ID NO: 93) (LCDR3) [Clone A-D5.16-DI]; or(z.1) the amino acid sequences GSGYTFTNYY (SEQ ID NO: 15) (HCDR1),INPVDGDTNYNPSFQG (SEQ ID NO: 91) (HCDR2), GGYTMD (SEQ ID NO: 16)(HCDR3), SSQSLLHSNGYTY (SEQ ID NO: 92) (LCDR1), KVSNRFS (SEQ ID NO: 85)(LCDR2) and NTHTPR (SEQ ID NO: 93) LCDR3) [Clone A-D5-DI].

The antibody molecule or antigen-binding portion of the invention(defined using the AHo definition) may comprise: an HCDR1 having theamino acid sequence GSGYTFTNYY (SEQ ID NO: 15) or GSGYSFTNYY (SEQ ID NO:86);

an HCDR2 having the amino acid sequence INPVDGDTNYNPSFQG (SEQ ID NO: 91)or INPVDGDTRYSPSFQG (SEQ ID NO: 100); and

an HCDR3 having the amino acid sequence GGYTMD (SEQ ID NO: 16), andoptionally further comprising:

an LCDR1 having the amino acid sequence SSQSLLHSNGYNY (SEQ ID NO: 89) orSSQSLLHSNGYTY (SEQ ID NO: 92) or SSQSLLHSAGYNY (SEQ ID NO: 108);

an LCDR2 having the amino acid sequence KVSNRLS (SEQ ID NO: 53) orKVSNRFS (SEQ ID NO: 85); and

an LCDR3 having the amino acid sequence NTHTPR (SEQ ID NO: 93).

The above antibody molecule or antigen-binding portion may alternativelydefined using the equivalent Unified CDR definitions as disclosedherein.

In particular embodiments of the invention, the antibody molecule orantigen-binding portion may comprise the six CDR sequences of CloneA-D5, Clone A-D5.4 or Clone A-D5.16 or Clone A-D5.16-DI or Clone A-D5-DIas defined above, or a suitable combination of the CDR sequences fromeach of these clones.

For example, in the antibody molecule or antigen-binding portion asdefined using the Unified CDR definition, the HCDR1 may have the aminoacid sequence: G-Y-T/S-F-T-N-Y-Y-I-F (SEQ ID NO: 27); the HCDR2 may havethe amino acid sequence: M-G-I-I-N-P-V-D-G-D-T-N/R-Y-N/S-P-S-F-Q-G (SEQID NO: 111); the HCDR3 may have the amino acid sequence: G-G-Y-T-M-D-R(SEQ ID NO: 51); the LCDR1 may have the amino acid sequence:R-S-S-Q-S-L-L-H-S-N/A-G-Y-N/T-Y-L-H (SEQ ID NO: 112); the LCDR2 may havethe amino acid sequence: K-V-S-N-R-L/F-S(SEQ ID NO: 113); and the LCDR3may have the amino acid sequence: F-Q-N-T-H-T-P-R-T (SEQ ID NO: 54).

Alternatively, in the antibody molecule or antigen-binding portion asdefined using the AHo definition, the HCDR1 may have the amino acidsequence: G-S-G-Y-T/S-F-T-N-Y-Y (SEQ ID NO: 30); the HCDR2 may have theamino acid sequence: I-N-P-V-D-G-D-T-N/R-Y-N/S-P-S-F-Q-G (SEQ ID NO:114); the HCDR3 may have the amino acid sequence: G-G-Y-T-M-D (SEQ IDNO: 16); the LCDR1 may have the amino acid sequence:S-S-Q-S-L-L-H-S-N/A-G-Y-N/T-Y (SEQ ID NO: 115); the LCDR2 may have theamino acid sequence: K-V-S-N-R-L/F-S(SEQ ID NO: 113); and the LCDR3 mayhave the amino acid sequence: N-T-H-T-P-R (SEQ ID NO: 93).

The antibody molecule or antigen-binding portion as defined herein maycomprise one or more substitutions, deletions and/or insertions whichremove a post-translational modification (PTM) site, for example aglycosylation site (N-linked or O-linked), a deamination site, aphosphorylation site or an isomerisation/fragmentation site.

More than 350 types of PTM are known. Key forms of PTM includephosphorylation, glycosylation (N- and O-linked), sumoylation,palmitoylation, acetylation, sulfation, myristoylation, prenylation andmethylation (of K and R residues). Statistical methods to identifyputative amino acid sites responsible for specific PTMs are well knownin the art (see Zhou et al., 2016, Nature Protocols 1: 1318-1321).Removal of such a site for example by substitution, deletion and/orinsertion and then optionally testing (experimentally and/ortheoretically) for (a) binding activity and/or (b) loss of the PTM iscontemplated.

For example, the VxP037 murine LCDR1 (as defined herein, i.e. the aminoacid sequence RSSQSLVHSNGNTYLH (SEQ ID NO: 9)) has been identified tohave a putative deamidation site at residue 10 (N) and/or 12 (N).Removal of either of both of these sites at equivalent positions in anLCDR1 of the invention, for example by conservative substitution (suchas to S, A, Q or D), is envisaged (as for example in clone A-D5.8, cloneA-D5 and other clones in Tables 3 and 4, or clones A-D5.11 to A-D5.18 inTable 5).

Similarly, the VxP037 murine LCDR3 (as defined herein, i.e. the aminoacid sequence SQNTHVPRT (SEQ ID NO: 11)) has been identified to have aputative deamidation site at residue 3 (N). Removal of this site at anequivalent position in an LCDR3 of the invention, for example byconservative or non-conservative substitution (such as to A, S, H, D, T,K, G, E, Q or R), is envisaged (as for example in clone F-E7 and otherclones in Tables 3 and 4).

Similarly, the VxP037 murine HCDR3 (as defined herein, i.e. the aminoacid sequence GGYTMDY (SEQ ID NO: 5)) has been identified to have aputative oxidation site at residue 5 (M). Removal of this site at anequivalent position in an HCDR3 of the invention, for example byconservative or non-conservative substitution (such as to P, A, T, S, L,F, W, V, I, Y or R), is envisaged (as for example in clone D-H3 andother clones in Tables 3 and 4).

The antibody molecule or antigen-binding portion thereof may be human,humanized or chimeric.

The antibody molecule or antigen-binding portion thereof may compriseone or more human variable domain framework scaffolds into which theCDRs have been inserted.

The antibody molecule or antigen-binding portion thereof may comprise anIGHV5-51 human germline scaffold into which the corresponding HCDRsequences have been inserted.

The antibody molecule or antigen-binding portion thereof may comprise anIGKV2-28 human germline scaffold into which the corresponding LCDRsequences have been inserted.

The antibody molecule or antigen-binding portion thereof may comprise animmunologically inert constant region.

The antibody molecule or antigen-binding portion thereof may be a Fabfragment, a F(ab)₂ fragment, an Fv fragment, a tetrameric antibody, atetravalent antibody, a multispecific antibody (for example, a bivalentantibody), a single domain antibody (for example, a shark antibody[V_(NAR) antibody], or a fragment thereof, or a camelid antibody [V_(H)Hantibody], or a fragment thereof), a monoclonal antibody or a fusionprotein. Antibody molecules and methods for their construction and useare described, in for example Holliger & Hudson (2005, NatureBiotechnol. 23(9): 1126-1136).

In another aspect of the invention, there is provided an immunoconjugatecomprising the antibody molecule or antigen-binding portion thereof ofthe invention as defined herein linked a therapeutic agent.

Examples of suitable therapeutic agents include cytotoxins,radioisotopes, chemotherapeutic agents, immunomodulatory agents,anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents,and cytostatic and cytolytic enzymes (for example RNAses). Furthertherapeutic agents include a therapeutic nucleic acid, such as a geneencoding an immunomodulatory agent, an anti-angiogenic agent, ananti-proliferative agent, or a pro-apoptotic agent. These drugdescriptors are not mutually exclusive, and thus a therapeutic agent maybe described using one or more of the above terms.

Examples of suitable therapeutic agents for use in immunoconjugatesinclude the taxanes, maytansines, CC-1065 and the duocarmycins, thecalicheamicins and other enediynes, and the auristatins. Other examplesinclude the anti-folates, vinca alkaloids, and the anthracyclines. Planttoxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes,photosensitizers may also be used in immunoconjugates. In addition,conjugates can be made using secondary carriers as the cytotoxic agent,such as liposomes or polymers, Suitable cytotoxins include an agent thatinhibits or prevents the function of cells and/or results in destructionof cells. Representative cytotoxins include antibiotics, inhibitors oftubulin polymerization, alkylating agents that bind to and disrupt DNA,and agents that disrupt protein synthesis or the function of essentialcellular proteins such as protein kinases, phosphatases, topoisomerases,enzymes, and cyclins.

Representative cytotoxins include, but are not limited to, doxorubicin,daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone,epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin,cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine,azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine,cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin,tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide,dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin,mechlorethamine, prednisone, procarbazine, methotrexate, flurouracils,etoposide, taxol, taxol analogs, platins such as cis-platin andcarbo-platin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin,epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen,idarubicin, dolastatins/auristatins, hemiasterlins, esperamicins andmaytansinoids.

Suitable immunomodulatory agents include anti-hormones that blockhormone action on tumors and immunosuppressive agents that suppresscytokine production, down-regulate self-antigen expression, or mask MHCantigens.

Also provided is a nucleic acid molecule encoding the antibody moleculeor antigen-binding portion thereof of the invention as defined herein.

Further provided is a vector comprising the nucleic acid molecule of theinvention as defined herein.

Also provided is a host cell comprising the nucleic acid molecule or thevector of the invention as defined herein.

In a further aspect there is provided a method of producing an anti-CD47antibody and/or an antigen-binding portion thereof, comprising culturingthe host cell of the invention under conditions that result inexpression and/or production of the antibody and/or the antigen-bindingportion thereof, and isolating the antibody and/or the antigen-bindingportion thereof from the host cell or culture.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising the antibody molecule or antigen-binding portionthereof of the invention as defined herein, or the nucleic acid moleculeof the invention as defined herein, or the vector of the invention asdefined herein.

Further provided is a method for enhancing an immune response in asubject, comprising administering an effective amount of the antibodymolecule or antigen-binding portion thereof of the invention as definedherein, or the immunoconjugate of the invention as defined herein, orthe nucleic acid molecule of the invention as defined herein, or thevector of the invention as defined herein, or the pharmaceuticalcomposition of the invention as defined herein.

In a further aspect there is provided a method for treating orpreventing cancer in a subject, comprising administering an effectiveamount of the antibody molecule or antigen-binding portion thereof ofthe invention as defined herein, or the immunoconjugate of the inventionas defined herein, or the nucleic acid molecule of the invention asdefined herein, or the vector of the invention as defined herein, or thepharmaceutical composition of the invention as defined herein.

The cancer may for example be selected from the group consisting of:pancreatic cancer, melanoma, breast cancer, lung cancer, bronchialcancer, colorectal cancer, prostate cancer, stomach cancer, ovariancancer, urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancerof hematological tissues.

The invention also provides an antibody molecule or antigen-bindingportion thereof of the invention as defined herein, or theimmunoconjugate of the invention as defined herein, or the nucleic acidmolecule of the invention as defined herein, or the vector of theinvention as defined herein, or the pharmaceutical composition of theinvention as defined herein, for use in the treatment of cancer.

In another aspect the invention provides the antibody molecule, orantigen-binding portion thereof, or the immunoconjugate, or the nucleicacid molecule, or the vector for use, or the method of treatment of theinvention as defined herein, for separate, sequential or simultaneoususe in a combination combined with a second therapeutic agent, forexample an anti-cancer agent.

In a further aspect there is provided the use of an antibody molecule orantigen-binding portion thereof of the invention as defined herein, oran immunoconjugate of the invention as defined herein, or a nucleic acidmolecule of the invention as defined herein, or a vector of theinvention as defined herein, or a pharmaceutical composition of theinvention as defined herein, in the manufacture of a medicament for thetreatment of cancer.

The invention also provides a method for treating or preventing anischemia-reperfusion injury, an autoimmune disease or an inflammatorydisease in a subject, comprising administering an effective amount ofthe antibody molecule or antigen-binding portion thereof as definedherein, or the immunoconjugate as defined here, or the nucleic acidmolecule as defined herein, or the vector as defined herein, or thepharmaceutical composition as defined herein.

The ischemia-reperfusion injury in all aspect may occur in organtransplantation, acute kidney injury, cardiopulmonary bypass surgery,pulmonary hypertension, sickle cell disease, myocardial infarction,stroke, surgical resections and reconstructive surgery, reattachment ofan appendage or other body part, skin grafting or trauma.

The autoimmune disease or inflammatory disease may be selected from thegroup consisting of: arthritis, multiple sclerosis, psoriasis, Crohn'sdisease, inflammatory bowel ‘disease, lupus, Grave's disease andHashimoto's thyroiditis, and ankylosing spondylitis.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of an ischemia-reperfusion injury, an autoimmune diseaseor an inflammatory disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of anischemia-reperfusion injury, an autoimmune disease or an inflammatorydisease.

The invention also provides a method for treating or preventing acardiovascular disease or a fibrotic disease in a subject, comprisingadministering an effective amount of the antibody molecule orantigen-binding portion thereof as defined herein, or theimmunoconjugate as defined here, or the nucleic acid molecule as definedherein, or the vector as defined herein, or the pharmaceuticalcomposition as defined herein.

Also provided is an antibody molecule or antigen-binding portion thereofas defined herein, or the immunoconjugate as defined herein, or thenucleic acid molecule as defined herein, or the vector as definedherein, or the pharmaceutical composition as defined herein, for use inthe treatment of a cardiovascular disease or a fibrotic disease.

Further provided is the use of an antibody molecule or antigen-bindingportion thereof as defined herein, or an immunoconjugate as definedherein, or a nucleic acid molecule as defined herein, or a vector asdefined herein, or a pharmaceutical composition as defined herein, inthe manufacture of a medicament for the treatment of a cardiovasculardisease or a fibrotic disease.

The cardiovascular disease in any aspect of the invention may forexample be coronary heart disease or atherosclerosis.

The fibrotic disease in any aspect of the invention may be selected fromthe group consisting of myocardial infarction, angina, osteoarthritis,pulmonary fibrosis, asthma, cystic fibrosis and bronchitis.

The pharmaceutical composition of the invention may comprise apharmaceutically acceptable excipient. A pharmaceutically acceptableexcipient may be a compound or a combination of compounds entering intoa pharmaceutical composition which does not provoke secondary reactionsand which allows, for example, facilitation of the administration of theanti-CD47 antibody molecule, an increase in its lifespan and/or in itsefficacy in the body or an increase in its solubility in solution. Thesepharmaceutically acceptable vehicles are well known and will be adaptedby the person skilled in the art as a function of the mode ofadministration of the anti-CD47 antibody molecule.

In some embodiments, the anti-CD47 antibody molecule may be provided ina lyophilised form for reconstitution prior to administration. Forexample, lyophilised antibody molecules may be re-constituted in sterilewater and mixed with saline prior to administration to an individual.

The anti-CD47 antibody molecules will usually be administered in theform of a pharmaceutical composition, which may comprise at least onecomponent in addition to the antibody molecule. Thus pharmaceuticalcompositions may comprise, in addition to the anti-CD47 antibodymolecule, a pharmaceutically acceptable excipient, carrier, buffer,stabilizer or other materials well known to those skilled in the art.Such materials should be non-toxic and should not interfere with theefficacy of the anti-CD47 antibody molecule. The precise nature of thecarrier or other material will depend on the route of administration,which may be by bolus, infusion, injection or any other suitable route,as discussed below.

For parenteral, for example sub-cutaneous or intra-venousadministration, e.g. by injection, the pharmaceutical compositioncomprising the anti-CD47 antibody molecule may be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles, such as Sodium Chloride Injection, Ringe's Injection,Lactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be employed as requiredincluding buffers such as phosphate, citrate and other organic acids;antioxidants, such as ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3′-pentanol; and m-cresol); low molecularweight polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagines, histidine,arginine, or lysine; monosaccharides, disaccharides and othercarbohydrates including glucose, mannose or dextrins; chelating agents,such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions, such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants, such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG).

A pharmaceutical composition comprising an anti-CD47 antibody moleculemay be administered alone or in combination with other treatments,either simultaneously or sequentially dependent upon the condition to betreated.

An anti-CD47 antibody molecule as described herein may be used in amethod of treatment of the human or animal body, including prophylacticor preventative treatment (e.g. treatment before the onset of acondition in an individual to reduce the risk of the condition occurringin the individual; delay its onset; or reduce its severity after onset).The method of treatment may comprise administering the anti-CD47antibody molecule to an individual in need thereof.

Administration is normally in a “therapeutically effective amount”, thisbeing sufficient to show benefit to a patient. Such benefit may be atleast amelioration of at least one symptom. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the composition, themethod of administration, the scheduling of administration and otherfactors known to medical practitioners. Prescription of treatment, e.g.decisions on dosage etc., is within the responsibility of generalpractitioners and other medical doctors and may depend on the severityof the symptoms and/or progression of a disease being treated.Appropriate doses of antibody molecules are well known in the art(Ledermann J. A. et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe K.D. et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922). Specific dosages may be indicated herein or in the Physician'sDesk Reference (2003) as appropriate for the type of medicament beingadministered may be used. A therapeutically effective amount or suitabledose of an antibody molecule may be determined by comparing its in vitroactivity and in vivo activity in an animal model. Methods forextrapolation of effective dosages in mice and other test animals tohumans are known. The precise dose will depend upon a number of factors,including whether the antibody is for prevention or for treatment, thesize and location of the area to be treated, the precise nature of theantibody (e.g. whole antibody, fragment) and the nature of anydetectable label or other molecule attached to the antibody.

A typical antibody dose will be in the range 100 μg to 1 g for systemicapplications, and 1 μg to 1 mg for topical applications. An initialhigher loading dose, followed by one or more lower doses, may beadministered. Typically, the antibody will be a whole antibody, e.g. theIgG1 or IgG4 isotype. This is a dose for a single treatment of an adultpatient, which may be proportionally adjusted for children and infants,and also adjusted for other antibody formats in proportion to molecularweight. Treatments may be repeated at daily, twice-weekly, weekly ormonthly intervals, at the discretion of the physician. The treatmentschedule for an individual may be dependent on the pharmacokinetic andpharmacodynamic properties of the antibody composition, the route ofadministration and the nature of the condition being treated.

Treatment may be periodic, and the period between administrations may beabout two weeks or more, e.g. about three weeks or more, about fourweeks or more, about once a month or more, about five weeks or more, orabout six weeks or more. For example, treatment may be every two to fourweeks or every four to eight weeks. Treatment may be given before,and/or after surgery, and/or may be administered or applied directly atthe anatomical site of surgical treatment or invasive procedure.Suitable formulations and routes of administration are described above.

In some embodiments, anti-CD47 antibody molecules as described hereinmay be administered as sub-cutaneous injections. Sub-cutaneousinjections may be administered using an auto-injector, for example forlong term prophylaxis/treatment.

In some preferred embodiments, the therapeutic effect of the anti-CD47antibody molecule may persist for several half-lives, depending on thedose. For example, the therapeutic effect of a single dose of theanti-CD47 antibody molecule may persist in an individual for 1 month ormore, 2 months or more, 3 months or more, 4 months or more, 5 months ormore, or 6 months or more.

The invention also provides a method of producing an antibody moleculewhich specifically binds to human CD47 and optionally also to cynomolgusmonkey CD47 and/or to mouse CD47, or an antigen-binding portion thereof,comprising the steps of:

(1) grafting anti-CD47 CDRs from a non-human source into a humanv-domain framework to produce a humanized anti-CD47 antibody molecule orantigen-binding portion thereof;

(2) generating a phage library of clones of the humanized anti-CD47antibody molecule or antigen-binding portion thereof comprising one ormore mutations in the CDRs;

(3) screening the phage library for binding to human CD47 and optionallyalso to cynomolgus monkey CD47 and/or to mouse CD47;

(4) selecting clones from the screening step (3) having bindingspecificity to human CD47 and optionally also to cynomolgus monkey CD47and/or to mouse CD47; and

(5) producing an antibody molecule which specifically binds to humanCD47 and optionally also to cynomolgus monkey CD47 and/or to mouse CD47,or an antigen-binding portion thereof from clones selected from step(4).

The method may comprise a further step of producing additional clonesbased on the clones selected in step (4), for example based on furtherexploratory mutagenesis at specific positions in the CDRs of the clonesselected in step (4), to enhance humanization, minimise human T cellepitope content and/or improve manufacturing properties in the antibodymolecule or antigen-binding portion thereof produced in step (5).

The method may comprise a further step of assessing immunogenicity ofone or more v-domains in the clones selected in step (4) or the antibodymolecule produced in step (5), and optionally generating one or morefurther mutations, for example in a CDR and framework region, to reduceimmunogenicity. Immunogenicity may be assessed by identifying thelocation of T cell epitopes, for example using in silico technologies asdescribed herein.

Refinements applicable to the above method are as described in Example 1below.

As used herein, the term “CD47” refers to Integrin Associated Protein(IAP) and variants thereof that retain at least part of the biologicalactivity of CD47. As used herein, CD47 includes all mammalian species ofnative sequence CD47, including human, rat, mouse and chicken. The term“CD47” is used to include variants, isoforms and species homologs ofhuman CD47. Antibodies of the invention may cross-react with CD47 fromspecies other than human, in particular CD47 from cynomolgus monkey(Macaca fascicularis). In certain embodiments, the antibodies may becompletely specific for human CD47 and may not exhibit non-humancross-reactivity.

As used herein, an “antagonist” as used in the context of the antibodyof the invention or an “anti-CD47 antagonist antibody” (interchangeablytermed “anti-CD47 antibody”) refers to an antibody which is able to bindto CD47 and inhibit CD47 biological activity and/or downstreampathway(s) mediated by CD47 signalling. An anti-CD47 antagonist antibodyencompasses antibodies that can block, antagonize, suppress or reduce(including significantly) CD47 biological activity, including downstreampathways mediated by CD47 signalling, such as receptor binding and/orelicitation of a cellular response to CD47. For the purposes of thepresent invention, it will be explicitly understood that the term“anti-CD47 antagonist antibody” encompass all the terms, titles, andfunctional states and characteristics whereby CD47 itself, and CD47biological activity (including but not limited to its ability to enhancethe activation of phagocytosis by cells of the myeloid lineage), or theconsequences of the activity or biological activity, are substantiallynullified, decreased, or neutralized in any meaningful degree. CD47“specifically binds” “specifically interacts”, “preferentially binds”,“binds” or “interacts” with CD47 if it binds with greater affinity,avidity, more readily and/or with greater duration than it binds toother receptors.

An “antibody molecule” is an immunoglobulin molecule capable of specificbinding to a target, such as a carbohydrate, polynucleotide, lipid,polypeptide, etc., through at least one antigen recognition site,located in the variable region of the immunoglobulin molecule. As usedherein, the term “antibody molecule” encompasses not only intactpolyclonal or monoclonal antibodies, but also any antigen bindingfragment (for example, an “antigen-binding portion”) or single chainthereof, fusion proteins comprising an antibody, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site including, for example without limitation, scFv, singledomain antibodies (for example, shark antibodies [V_(NAR) antibodies],or a fragment thereof, and camelid antibodies [V_(H)H antibodies], or afragments thereof), maxibodies, minibodies, intrabodies, diabodies,triabodies, tetrabodies, and bis-scFv.

An “antibody molecule” encompasses an antibody of any class, such asIgG, IgA, or IgM (or sub-class thereof), and the antibody need not be ofany particular class. Depending on the antibody amino acid sequence ofthe constant region of its heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, and several of these may be further dividedinto subclasses (isotypes), for example IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2. The heavy-chain constant regions that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu, respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “antigen binding portion” of an antibody molecule, as usedherein, refers to one or more fragments of an intact antibody thatretain the ability to specifically bind to CD47. Antigen bindingfunctions of an antibody molecule can be performed by fragments of anintact antibody. Examples of binding fragments encompassed within theterm “antigen binding portion” of an antibody molecule include Fab;Fab′; F(ab′)2; an Fd fragment consisting of the VH and CH1 domains; anFv fragment consisting of the VL and VH domains of a single arm of anantibody; a single domain antibody (dAb) fragment, and an isolatedcomplementarity determining region (CDR).

The term “Fc region” is used to define a C-terminal region of animmunoglobulin heavy chain. The “Fc region” may be a native sequence Fcregion or a variant Fc region. Although the boundaries of the Fc regionof an immunoglobulin heavy chain might vary, the human IgG heavy chainFc region is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. Thenumbering of the residues in the Fc region is that of the EU index as inKabat. The Fc region of an immunoglobulin generally comprises twoconstant domains, CH2 and CH3. As is known in the art, an Fc region canbe present in dimer or monomeric form.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FRs) connected by three complementarity determining regions(CDRs) also known as hypervariable regions, contribute to the formationof the antigen binding site of antibodies. When choosing FR to flankCDRs, for example when humanizing or optimizing an antibody, FRs fromantibodies which contain CDR sequences in the same canonical class arepreferred.

The CDR definitions used in the present application combine the domainsused in the many disparate, often conflicting schemes that have beencreated in the field, which are based on the combination ofimmunoglobulin repertoire analyses and structural analyses of antibodiesin isolation and in their co-crystals with antigens (see review bySwindells et al., 2016, abYsis: Integrated Antibody Sequence andStructure-Management, Analysis, and Prediction. J Mol Biol. [PMID:27561707; Epub 22 Aug. 2016]). The CDR definition used herein (a“Unified” definition) incorporates the lessons of all such priorinsights and includes all appropriate loop positions required to samplethe full residue landscape that potentially mediates target-bindingcomplementarity.

Table 1 shows the amino acid sequences of the VxP037 murine anti-CD47antibody CDRs as defined herein (a “Unified” scheme), in comparison towell-known alternative systems for defining the same CDRs.

As used herein the term “conservative substitution” refers toreplacement of an amino acid with another amino acid which does notsignificantly deleteriously change the functional activity. A preferredexample of a “conservative substitution” is the replacement of one aminoacid with another amino acid which has a value ≥0 in the followingBLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89:10915-10919):

A R N D C Q E G H I L K M F P S T W Y V A 4 −1 −2 −2  0 −1 −1  0 −2 −1−1 −1 −1 −2 −1  1  0 −3 −2  0 R −1  5  0 −2 −3  1  0 −2  0 −3 −2  2 −1−3 −2 −1 −1 −3 −2 −3 N −2  0  6  1 −3  0  0  0  1 −3 −3  0 −2 −3 −2  1 0 −4 −2 −3 D −2 −2  1  6 −3  0  2 −1 −1 −3 −4 −1 −3 −3 −1  0 −1 −4 −3−3 C  0 −3 −3 −3  9 −3 −4 −3 −3 −1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1  1 0  0 −3  5  2 −2  0 −3 −2  1  0 −3 −1  0 −1 −2 −1 −2 E −1  0  0  2 −4 2  5 −2  0 −3 −3  1 −2 −3 −1  0 −1 −3 −2 −2 G  0 −2  0 −1 −3 −2 −2  6−2 −4 −4 −2 −3 −3 −2  0 −2 −2 −3 −3 H −2  0  1 −1 −3  0  0 −2  8 −3 −3−1 −2 −1 −2 −1 −2 −2 2 −3 I −1 −3 −3 −3 −1 −3 −3 −4 −3  4  2 −3  1  0 −3−2 −1 −3 −1  3 L −1 −2 −3 −4 −1 −2 −3 −4 −3  2  4 −2  2  0 −3 −2 −1 −2−1  1 K −1  2  0 −1 −3  1  1 −2 −1 −3 −2  5 −1 −3 −1  0 −1 −3 −2 −2 M −1−1 −2 −3 −1  0 −2 −3 −2  1  2 −1  5  0 −2 −1 −1 −1 −1  1 F −2 −3 −3 −3−2 −3 −3 −3 −1  0  0 −3  0  6 −4 −2 −2  1  3 −1 P −1 −2 −2 −1 −3 −1 −1−2 −2 −3 −3 −1 −2 −4  7 −1 −1 −4 −3 −2 S  1 −1  1  0 −1  0  0  0 −1 −2−2  0 −1 −2 −1  4  1 −3 −2 −2 T  0 −1  0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1−2 −1  1  5 −2 −2  0 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1  1 −4 −3−2 11  2 −3 Y −2 −2 −2 −3 −2 −1 −2 −3  2 −1 −1 −2 −1  3 −3 −2 −2  2  7−1 V  0 −3 −3 −3 −1 −2 −2 −3 −3  3  1 −2  1 −1 −2 −2  0 −3 −1   4.

The term “monoclonal antibody” (Mab) refers to an antibody, orantigen-binding portion thereof, that is derived from a single copy orclone, including for example any eukaryotic, prokaryotic, or phageclone, and not the method by which it is produced. Preferably, amonoclonal antibody of the invention exists in a homogeneous orsubstantially homogeneous population.

A “humanized” antibody molecule refers to a form of non-human (forexample, murine) antibody molecules, or antigen-binding portion thereof,that are chimeric immunoglobulins, immunoglobulin chains, or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-bindingsubsequences of antibodies) that contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies may be humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity.

“Human antibody or fully human antibody” refers to an antibody molecule,or antigen-binding portion thereof, derived from transgenic micecarrying human antibody genes or from human cells.

The term “chimeric antibody” is intended to refer to an antibodymolecule, or antigen-binding portion thereof, in which the variableregion sequences are derived from one species and the constant regionsequences are derived from another species, such as an antibody moleculein which the variable region sequences are derived from a mouse antibodyand the constant region sequences are derived from a human antibody.

“Antibody-drug conjugate” and “immunoconjugate” refer to an antibodymolecule, or antigen-binding portion thereof, including antibodyderivatives that binds to CD47 and is conjugated to cytotoxic,cytostatic and/or therapeutic agents.

Antibody molecules of the invention, or antigen-binding portion thereof,can be produced using techniques well known in the art, for examplerecombinant technologies, phage display technologies, synthetictechnologies or combinations of such technologies or other technologiesreadily known in the art.

The term “epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody molecule, or antigen-bindingportion thereof, at one or more of the antibody molecule'santigen-binding regions. Epitopes can consist of defined regions ofprimary secondary or tertiary protein structure and includescombinations of secondary structural units or structural domains of thetarget recognised by the antigen binding regions of the antibody, orantigen-binding portion thereof. Epitopes can likewise consist of adefined chemically active surface grouping of molecules such as aminoacids or sugar side chains and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.The term “antigenic epitope” as used herein, is defined as a portion ofa polypeptide to which an antibody molecule can specifically bind asdetermined by any method well known in the art, for example, byconventional immunoassays, antibody competitive binding assays or byx-ray crystallography or related structural determination methods (forexample NMR).

The term “binding affinity” or “KD” refers to the dissociation rate of aparticular antigen-antibody interaction. The KD is the ratio of the rateof dissociation, also called the “off-rate (k_(off))”, to theassociation rate, or “on-rate (k_(on))”. Thus, K_(D) equalsk_(off)/k_(on) and is expressed as a molar concentration (M). It followsthat the smaller the K_(D), the stronger the affinity of binding.Therefore, a K_(D) of 1 μM indicates weak binding affinity compared to aK_(D) of 1 nM. KD values for antibodies can be determined using methodswell established in the art. One method for determining the KD of anantibody is by using surface plasmon resonance (SPR), typically using abiosensor system such as a Biacore® system.

The term “potency” is a measurement of biological activity and may bedesignated as IC₅₀, or effective concentration of an antibody orantibody drug conjugate to the antigen CD47 to inhibit 50% of activitymeasured in a CD47 activity assay as described herein.

The phrase “effective amount” or “therapeutically effective amount” asused herein refers to an amount necessary (at dosages and for periods oftime and for the means of administration) to achieve the desiredtherapeutic result. An effective amount is at least the minimal amount,but less than a toxic amount, of an active agent which is necessary toimpart therapeutic benefit to a subject.

The term “inhibit” or “neutralize” as used herein with respect tobioactivity of an antibody molecule of the invention means the abilityof the antibody to substantially antagonize, prohibit, prevent,restrain, slow, disrupt, eliminate, stop, reduce or reverse for exampleprogression or severity of that which is being inhibited including, butnot limited to, a biological activity or binding interaction of theantibody molecule to CD47.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

The term “treating”, as used herein, unless otherwise indicated, meansreversing, alleviating, inhibiting the progress of, delaying theprogression of, delaying the onset of, or preventing the disorder orcondition to which such term applies, or one or more symptoms of suchdisorder or condition. The term “treatment”, as used herein, unlessotherwise indicated, refers to the act of treating as defined above. Theterm “treating” also includes adjuvant and neoadjuvant treatment of asubject. For the avoidance of doubt, reference herein to “treatment”includes reference to curative, palliative and prophylactic treatment.For the avoidance of doubt, references herein to “treatment” alsoinclude references to curative, palliative and prophylactic treatment.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Any example(s) following the term “e.g.” or “forexample” is not meant to be exhaustive or limiting.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art.

Particular non-limiting embodiments of the present invention will now bedescribed with reference to accompanying drawings.

Example 1. Generation and Characterisation of Optimized Anti-CD47Therapeutic Antibodies

Introduction

In this example, we successfully generate a panel of antagonistic,optimized anti-CD47 antibodies. These anti-CD47 antibodies are wellexpressed, biophysically stable, highly soluble and of maximizedidentity to preferred human germlines.

Materials and Methods

IgG Cloning, Transient Expression, Purification

Antibody v-domain encoding DNA sequences were cloned viarestriction-ligation cloning into separate IgG heavy and light-chainexpression cassettes in separate plasmid vectors. Antibodies wereexpressed in two forms of engineered human IgG: IgG4 with the S228Pmutation to stabilise the IgG4 hinge, and IgG1null—IgG1 with the lowerhinge mutations L234A/L235A/G237A, which minimise Fcγ receptor-driveneffector functions. IgGs were expressed in HEK-293expi cells aftertransient transfection with endotoxin-free IgG expression plasmidpreparations, per manufacturer's protocols. IgGs were purified using asingle-step protocol: Conditioned media were loaded (neat) onto a 1 mlProA sepharose column, pre-equilibrated in PBS pH7.4. The column waswashed with 5 column volumes of PBS pH7.4, before the protein was elutedwith 100 mM glycine, pH 2.7 and subjected to dialysis in PBS pH 7.4using 30 kDa cutoff dialysis membrane.

IgG Titration Binding ELISAs

To coat Greiner Bio-One High bind ELISA plates, target proteins werediluted to 1 μg/ml in carbonate buffer and added at 100 μl per well, at4° C., o/n. Coated plates were washed 3× with PBS pH7.4, blocked with 1%BSA in PBS (380 μl/well) for 1 hr at RT, then washed 3× with PBS-Tween20 (PBST). CD47 antibodies (100 μl/well; diluted in PBST) were thenadded and then incubated 1 hr at RT. Plates were then washed 3× withPBST and goat anti-human kappa chain-HRP added (100 μl/well) at RT, for1 hr. Plates were then washed 3× with PBST and twice with PBS before theaddition of 100 μl TMB per well. Reactions were stopped by adding 100 μl2M H₂SO₄/well and OD was read on a plate reader at 450 nm.

Anti-CD47 antibodies were tested for polyreactivity by ELISA. Purified,recombinant, target and non-target antigens were coated in 96-well Nuncmaxisorp plates at 100 ng per well in carbonate buffer, at 4° C.overnight. Plates were then washed 3× with PBS, blocked with 1% BSA inPBS, then washed 3× with PBS-Tween20. A dilution series of primaryantibodies was then applied, plates were washed 3× with PBS-Tween20followed by application of goat anti-human kappa chain-HRP 1:4,000secondary antibody. Wells were then washed 3× with PBS-Tween20 and 2×with PBS, 100 μl TMB peroxidase substrate was added per well, thereaction was stopped by adding 100 μl 2M H₂SO₄ and absorbances were readat 450 nm. IgG binding analysis via ELISA on negatively chargedbiomolecular surfaces were performed as previously described (seeMouquet et al., 2010, Nature 467: 591-595).

CD47 Library Generation and Selection

The CD47 scFv repertoire was assembled by mass oligo synthesis and PCR.The amplified scFv repertoire was then cloned via restriction-ligationinto a phagemid vector, transformed into E. coli TG-1 cells, and thephage repertoire rescued essentially as previously described in detail(Finlay et al., 2011, Methods Mol Biol 681: 383-401).

Phage selections were performed by coating streptavidin magneticmicrobeads with CD47-Fc protein (either human or cyno), washing thebeads thrice with PBS and resuspending in PBS pH7.4 plus 5% skim milkprotein (MPBS). These beads were coated at 200 nM target protein inround 1 of selection, followed by 100, 50 and 10 nM in subsequentrounds.

CD47-SIRPα Binding Competition Assay

A competition ELISA assay was established to examine the capacity ofoptimized leads to block the binding interaction of CD47 with SIRPα. Tocoat Greiner Bio-One High bind ELISA plates, 10 μg/ml human SIRPα-Fc incarbonate coating buffer was added at 100 μl per well, at 4° C., o/n.Coated plates were washed 3× with PBS pH7.4, blocked with 1% BSA in PBS(380 μl/well) for 1 hr at RT, then washed 3× with PBS-Tween 20 (PBST).Biotinylated human, mouse or cyno CD47-Fc was then added at 0.2 μg/ml inPBS, 100 μl per well, at room temperature for 60 minutes with or withoutthe addition of competing IgGs. Plates were then washed 3× with PBST andStreptavidin-HRP added (100 μl/well) at room temperature, for 1 hr.Plates were then washed 3× with PBST and twice with PBS before theaddition of 100 μl TMB per well. Reactions were stopped by adding 100 μl2M H₂SO₄/well and OD was read on a plate reader at 450 nm.

Antibody v-Domain T Cell Epitope Content: In Silico Analyses

In silico technologies (Abzena, Ltd.), which are based on identifyingthe location of T cell epitopes in therapeutic antibodies and proteins,were used for assessing potential immunogenicity in antibody v-domains.iTope™ was used to analyse the VL and VH sequences of key leads forpeptides with promiscuous high affinity binding to human MHC class II.Promiscuous high affinity MHC class II binding peptides are thought tocorrelate with the presence of T cell epitopes that are high riskindicators for clinical immunogenicity of drug proteins. The iTope™software predicts favourable interactions between amino acid side chainsof a peptide and specific binding pockets (in particular pocketpositions; p1, p4, p6, p7 and p9) within the open-ended binding groovesof 34 human MHC class II alleles. These alleles represent the mostcommon HLA-DR alleles found world-wide with no weighting attributed tothose found most prevalently in any particular ethnic population. Twentyof the alleles contain the ‘open’ p1 configuration and 14 contain the‘closed’ configuration where glycine at position 83 is replaced by avaline. The location of key binding residues is achieved by the insilico generation of 9mer peptides that overlap by eight amino acidsspanning the test protein sequence. This process successfullydiscriminates with high accuracy between peptides that either bind or donot bind MHC class II molecules.

In addition, the sequences were analysed using TCED™ (T Cell EpitopeDatabase™) search for matches to T cell epitopes previously identifiedby in vitro human T cell epitope mapping analyses of other proteinsequences. The TCED™ is used to search any test sequence against a large(>10,000 peptides) database of peptides derived from unrelated proteinand antibody sequences.

Cancer Cell Phagocytosis Analysis

Human peripheral blood mononuclear cells (PBMCs) were isolated fromwhole blood by density gradient centrifugation. CD14+ PBMCs weresubsequently isolated via magnetic cell isolation using CD14 microbeads.In parallel, HL-60 cells were labelled using a green CFSE(carboxy-fluorescein diacetate, succinimidyl ester) cell tracer dye. Atotal of 1.25×10⁶ labelled HL-60 cells were pre-incubated in thepresence of anti-CD47 antibodies in 24 well plates for 1 hour at 37° C.in a humidified atmosphere containing 5% CO₂. Following incubation,5×10⁵ CD14 positive cells were added to each well and incubated for afurther hour under the same culture conditions. Cells were harvested byvigorous pipetting, fixed using ice-cold 4% paraformaldehyde for 10minutes and then blocked with an Fc receptor binding inhibitormonoclonal antibody for 10 minutes. Following the blocking step cellswere incubated with an Alexa Fluor 647 (AF647) conjugated anti-humanCD14 antibody at room temperature for 30 minutes and fixed a furthertime in 4% paraformaldehyde for 5 minutes.

Cells were analysed on the BD Fortessa flow cytometer recording sidescatter and forward scatter properties along with CFSE and AF647fluorescence intensity data. Data was captured until at least 1×10⁴AF647 positive events were recorded. Data were analysed post-acquisitionusing FlowJo software (version 10.4.2). Briefly, cell debris was gatedout by scatter properties (SSC-Area by FSC-Area). Single cells were alsogated for by SSC-Area by SSC-Height and then by FSC-Area by FSC-Height.From the remaining single cell population, CFSE and CD14 doublepositives cells were gated using a quadrant gate placed based on thepopulation of CD14 positive cells in the vehicle treated test. Thepercentage of CFSE positive cells from the CD14 positive population wascalculated and plotted using GraphPad Prism software (version 7.0a).

Results and Discussion

CDR Grafting onto Preferred Human Germline v-Genes

The CDRs of an antagonistic murine anti-CD47 IgG VP037 (mVH/mVL; seeWO2014/093678 and Table 2) were initially introduced to human germlineimmunoglobulin v-domain framework sequence scaffolds using CDR grafting.To bias our engineering efforts towards final lead therapeutic IgGcompounds with optimal drug-like properties, we chose to graft the CDRsof the parental antibody onto “preferred” germline scaffolds IGHV5-51and IGKV2-28, which are known to have good solubility and are used athigh frequency in the expressed human antibody repertoire.

Those scaffolds and grafted CDR definitions are outlined in Table 2. Theheavy and light chain sequences for murine anti-CD47 antibody are alsoshown in Table 2. While this process of CDR grafting is well known, itis still problematic to predict whether a given set of human v-domainsequences will act as suitable acceptor frameworks for non-human CDRgrafting. The use of unsuitable frameworks can lead to the loss oftarget binding function, protein stability issues or even impairedexpression of the final IgG. The IGHV5-51/IGKV2-28 graft was thereforetaken forward as the template for CDR mutagenesis and selection ofimproved clones.

Library Generation and Screening

The CDR-grafted IGHV5-51/IGKV2-28 v-domain sequences were combined intoa VL-VH scFv format and a mutagenesis library cassette was generated bymass oligo synthesis and assembly. The final scFv library was ligatedinto a phage display vector and transformed into E. coli viaelectroporation to generate 1.3×10⁹ independent clones. Library buildquality was verified by sequencing 96 clones. This sequencing datashowed that the positions encoding either the murine or human germlineresidue at each position of variance had been effectively sampled at afrequency of approximately 50%. Libraries were rescued using helperphage M13 and selections performed on biotinylated human, mouse andcynomolgus monkey CD47-Fc proteins in three separate branches A, B andC.

Post-selection screening (FIG. 1) and DNA sequencing revealed thepresence of 854 unique, human and mouse CD47-binding scFv clones withsignificantly increased human content within the CDRs, while theframework sequences remained fully germline. Amongst these 854 clones,germ-lining mutations were observed in all CDRs (Table 3). Lead cloneswere ranked based on level of CDR germ-lining versus ELISA signal forbinding to both human and mouse CD47-Fc (FIG. 1). The v-domains of the 4top clones from this ranking, plus a 5^(th) clone (‘VH-A1/VL-B1’) thatcombined the two most humanized observed heavy and light chain v-domainswere then sub-cloned into IgG expression vectors for further testing asbelow (Table 4).

While germ-lining mutations were observed in all CDRs for the leadclones derived directly from library selections, it remained possiblethat sequence analyses might allow further clones to be designed to havemaximal humanization. The 854 sequence-unique hits with binding signalsagainst human and mouse protein were therefore used to analyse theretention frequency for murine amino acids in the CDRs of thisfunctionally characterized population. Positional amino acid retentionfrequency was expressed as a percentage found in the V_(H) and V_(L)domains (FIG. 2A&B). Murine residues with RF<75% were regarded aspositions that are possibly not essential to the target-binding paratopeand are likely to be open to germ-lining, in a series of combinatorialdesigns.

A design containing only those murine residues with RF>75% wasdesignated “MH” (MH=Maximally Humanized). Another designer clone(‘TTP’=Total Theoretically Possible) was also created that combined the5 most humanized CDRs observed in the population. The MH and TTP cloneswere generated by gene synthesis and (along with the 4 library-derivedclones outlined above and positive control mVH/mVL and negative controlisotype-matched non-CD47-reactive v-domains), cloned into humanexpression vectors for production as IgG1null and IgG4(S228P). All IgGswere readily expressed and purified from transient transfections ofHEK-293 cells.

Lead IgG Specificity and Potency Characteristics

The purified IgGs described above were then tested for binding to human,mouse and cyno CD47-Fc in direct titration ELISA format (FIG. 3).Surprisingly, this analysis demonstrated that while clones MH, A-D5, andD-H3 retained binding affinity for all 3 orthologues of CD47, two clonesshowed reduced binding to mouse CD47 (G-B6 and F-E7), one clone(VHA-1/VL-B1) maintained comparable binding to both human and mouseCD47, but had lost cross-reactivity to mouse CD47, and one clone (TTP)had lost almost all binding function.

In a CD47-SIRPα blockade assay (FIG. 4), A-D5, G-B6, F-E7 and D-H3 allexhibited concentration-dependent blockade of human, mouse and cyno CD47interaction with SIRPα-Fc in similar concentration ranges as the mVH/mVLIgG1 antibody. Notably, the VH-A1/VL-B1 IgG1 exhibited potent blockadeof human and cyno CD47, but no blockade of mouse CD47. Interestingly,clone MH, despite having demonstrated binding to all 3 orthologues ofCD47 in ELISA, showed no blockade signal in any assay. Clone TTP wasalso negative in all blockade assays.

To ensure that that lead clones had not suffered from loss of targetspecificity during the mutation and reselection process; lead andcontrol IgG1 clones were tested for binding to a panel of 14 purifiedhuman proteins from the immunoglobulin superfamily (FIG. 5). All fiveIgGs exhibited binding signals at 1 μg/ml to CD47-Fc (human OD450nm>2.0, cyno >2.0, mouse >1.25), and no detectable binding (OD450nm<0.1) against any other protein. One notable exception was theVH-A1/VI-B1 clone, which again showed strong binding to human and cynoCD47, but no signal against mouse CD47.

Flow Cytometric Analyses of Lead IgG Binding Specificity at the CellMembrane

Antibodies to CD47 were analysed for concentration-dependent binding atthe cell surface via flow cytometry. CHO-K1 cells were stablytransfected with either human, mouse or cyno CD47 full-length cDNAs.Anti-CD47 IgGs mVH/mVL, VH-A1/VL-B1, A-D5, G-B6, F-E7 and D-H3, and anisotype control IgG1 were then all tested in IgG1null and IgG4(S228P)formats, alongside a commercial mouse anti-human CD47 monoclonal MS1991,over a concentration range of 100,000-24 ng/ml for binding to human,cyno or wild type control (‘wt’, i.e. untransfected) CHO-K1. All IgGsother than the isotype control showed concentration-dependent binding tohuman and cyno CD47+ cells, with a maximum MFI in each casebeing >10-fold higher than observed signals for binding to untransfectedCHO-K1 (FIG. 6). Measurable binding to CHO-K1 wt cells was observed forall clones other than VH-A1/VL-B1, but only at high antibodyconcentrations.

The mVH/mVL IgGs, however, showed the strongest reactivity with >10-foldhigher signal than the ‘no antibody’ negative control at concentrationsas low as 24 ng/ml. This background binding may be indicative of theoriginal mouse antibody VxP037 having not just cross-reactivity to mouseCD47, but also hamster. The minimisation of this cross-reactivity tohamster CD47 is preferable for a therapeutic protein, as antibodies willtypically be produced in CHO cell culture for clinical use. High IgGbinding affinity for hamster CD47 protein may potentially lead to asignificant increase in the content of unwanted co-purified CD47 hostcell protein in production runs, which must be removed from the drugproduct to avoid immunogenicity in patients.

To examine the binding of the lead IgGs to CD47+ human cancer cells,HL60 cells (derived from human acute myeloid leukaemia) were also usedin flow cytometry analyses, as above. All antibodies other than theIsotype control IgGs showed strong, concentration-dependent binding tothe HL60 cells (FIG. 7).

Lead IgG Analyses in ‘Developability’ ELISA Assays

It is known in the art that the binding of IgGs intended for therapeuticuse to several indicative biological substrates is an indicator of highrisk for poor performance in patients due to poor bioavailability andshort in vivo half-life. Three such biological substrates are Insulin,dsDNA and ssDNA. These three substrates were therefore used to coatELISA plates and examine the binding of the IgG1null versions of theoptimised lead antibodies. Binding signals for these human IgG-basedantibodies was compared to ‘positive control’ human IgG antibodies thathave been found to have polyreactivity and poor performance, whichstopped their progress in clinical trials (Bococizumab and Briakinumabhuman IgG1 analogues). For a negative control human IgG1 antibody, anIgG1 Ustekinumab analogue was used as it reacts with the sametherapeutic target as Briakinumab, but has longer pK and wassuccessfully approved as a therapeutic product. In the ELISA analysesshown in FIG. 8, the positive control antibodies exhibited the expectedstrong reactivity to all 3 substrates, while the negative control showedlow reactivity. Importantly, all of the IgG1 null lead proteins testedshowed binding ≤the negative control against all 3 substrates. Thisfinding underlined the maintenance of highly specific, target-drivenbinding in the optimised clones A-D5, G-B6, D-H3 and VH-A1/VL-B1.

Analyses of Designer IgGs Based on the Lead Clone A-D5

As described above, clone A-D5 had proven to have highly specificbinding to human, cyno and mouse CD47, low off-target binding potential,improved neutralisation of mouse CD47, reduced background binding to CHOcells and multiple human germlining mutations in the CDRs. As alibrary-derived clone, however, the A-D5 sequence still retained anumber of non-germline (mouse-derived) residues that were suggested tobe potentially superfluous by the data found in FIGS. 2A and 2B. TheA-D5 sequence and all other library-derived clones also retained an ‘NG’motif in the CDR-L1 that posed high risk for deamidation, as it wasfound to exhibit high solvent exposure at the apex of the long,flexible, IGKV2-28 germline-template CDR-L1 loop. In an attempt tosimultaneously maximise human germline sequence in the variable domainsof A-D5 and to minimise high-risk deamidation motif content in theprotein, a series of designer clones were created. This effort wascarried out in two phases, with the A-D5.1 to A-D5.10 clones from thefirst phase containing the CDR sequences outlined in Table 4. Theseclones were expressed and purified in IgG1null format and examined fortarget binding by ELISA on all CD47 orthologs (FIG. 9A, B, C) andneutralisation of CD47-SIRPα interaction for all orthologs (FIG. 10A, B,C). In this phase, it was found that the human germline and theseimprovements could be combined with a moderate loss of potency. Inaddition, the initial mutations attempting to remove the ‘NG’ motif inthe CDR-L1 (via conservative substitution of N to Q) were successful,albeit with associated reductions in potency in both target-bindingELISAs and neutralisation of CD47-SIRPα interaction (FIG. 10A, B, C).

In the second phase, a series of further mutants were designed on thehighest-performing mutant from phase 1: A-D5.4 (Table 5). These 9mutants sampled further humanization of the CDR-H2 of A-D.4, with orwithout also replacing the ‘N’ in the ‘NG’ deamidation risk motif withconservative and non-conservative mutations such as S, G, A and T, orreplacing the ‘G’ residue with A. These clones were again expressed andpurified in IgG1null format and examined for target binding by ELISA onall CD47 orthologs (FIG. 11A, B, C) and neutralisation of CD47-SIRPαinteraction for all orthologs (FIG. 12A, B, C). In this phase, it wasfound that the human germline content of the CDR-H2 could be raised by 2further residues, without loss of potency in comparison to clone A-D5.In addition, the mutations attempting to remove the ‘NG’ motif in theCDR-L1 via substitution of N were successful, leading to identificationof the clone A-D5.16 which contained the non-conservative N to Amutation, plus the maximally humanized CDR-H2, with only minor(approximately 3-fold) reductions in potency in either target-bindingELISAs or neutralisation of CD47-SIRPα, versus both A-D5 and mVL/mVH.The CDR-L1 sequence of A-D.16 ‘RSSQSLLHSAGYNYLH’ (SEQ ID NO: 82) (andclones A-D5.14, A-D5.15, A-D5.17 and A-D5.28) contained non-germlineresidues at only two positions (underlined) and achieved an optimizedbalance between maximum human germline content and maximum stabilitycharacteristics.

The A-D5 derivatives A-D5.4 and A-D5.16 were subsequently also tested inIgG1null format for maintenance of binding specificity to ensure thatthere had been no loss of target specificity during the mutation andreselection process; both clones were tested for binding to a panel of14 purified human proteins from the immunoglobulin superfamily (FIG.13). Both IgGs exhibited binding signals at 10 μg/ml to CD47-Fc (human,cyno and mouse all >2.0 OD450 nm), and no detectable binding (OD450nm<0.1) against any other protein. In the ‘Developability’ ELISAanalyses shown in FIG. 14, the positive control antibodies exhibited theexpected strong reactivity to all 3 substrates, while the negativecontrol showed low reactivity. Importantly, both A-D5.4 and A-D5.16IgG1null lead proteins showed binding≤the negative control against all 3substrates. This finding underlined the maintenance of highly specific,target-driven binding in the optimised clones A-D5, A-D5.4 and A-D5.16.

Finally, the A-D5 derivative mutant A-D5.4 and A-D5.16 were examined fortheir binding to wild type CHO (FIG. 15) and HL60 (FIG. 16) cells byflow cytometry. These analyses confirmed that the original murinev-domains of clone mVH/mVL in either IgG1null or IgG4 format drivestrong, concentration-dependent binding to CHO cells, whereas clonesA-D5, A-D5.4 and A-D5.16 mediate little or no binding signal in eitherIgG format (FIG. 15). In contrast, clones mVH/mVL, A-D5, A-D5.4 andA-D5.16 all demonstrated strong binding to human CD47+HL60 cells (FIG.16), demonstrating that human CD47 binding at the cell membrane hadindeed been retained in our optimized clones, but hamster CD47reactivity had been ameliorated.

Antibody v-Domain T Cell Epitope Analyses

In silico technologies (Abzena, Ltd.), which are based on identifyingthe location of T cell epitopes in therapeutic antibodies and proteins,were used for assessing the immunogenicity of both the mVH/mVL and leadantibody v-domains. Analysis of the v-domain sequences was performedwith overlapping 9mer peptides (with each overlapping the last peptideby 8 residues) which were tested against each of the 34 MHC class IIallotypes. Each 9mer was scored based on the potential ‘fit’ andinteractions with the MHC class II molecules. The peptide scorescalculated by the software lie between 0 and 1. Peptides that produced ahigh mean binding score (>0.55 in the iTope™ scoring function) werehighlighted and, if >50% of the MHC class II binding peptides (i.e. 17out of 34 alleles) had a high binding affinity (score >0.6), suchpeptides were defined as ‘high affinity’ MHC class II binding peptideswhich are considered a high risk for containing CD4+ T cell epitopes.Low affinity MHC class II binding peptides bind a high number of alleles(>50%) with a binding score >0.55 (but without a majority >0.6). Furtheranalysis of the sequences was performed using the TCED™. The sequenceswere used to interrogate the TCED™ by BLAST search in order to identifyany high sequence homology between peptides (T cell epitopes) fromunrelated proteins/antibodies that stimulated T cell responses inprevious in vitro T cell epitope mapping studies performed at AbzenaLtd.

Peptides were grouped into four classes: High Affinity Foreign(‘HAF’—high immunogenicity risk), Low Affinity Foreign (‘LAF’—lowerimmunogenicity risk), TCED+(previously identified epitope in TCED™database), and Germline Epitope (‘GE’—human germline peptide sequencewith high MHC Class II binding affinity). Germline Epitope 9mer peptidesare unlikely to have immunogenic potential due to T cell tolerance (i.e.these peptides are recognised as ‘self’ in the host), as validated byprevious studies with a wide range of germline peptides. Importantly,such germline v-domain epitopes (aided further by similar sequences inthe human antibody constant regions) also compete for MHC Class IIoccupancy at the membrane of antigen presenting cells, reducing the riskof foreign peptide presentation being sufficient to achieve the‘activation threshold’ required for T cell stimulation. High GE contentis therefore a beneficial quality in clinical development of an antibodytherapeutic.

As shown in FIG. 17, key lead v-domains exhibited significant beneficialchanges in peptide epitope content in comparison to mVH/mVL. As thev-domain engineering process undertaken here had successfully selectedfor antibodies that maintained anti-CD47 potency without the need forany murine residues being included in the frameworks (Table 2), multipleHAF and LAF epitopes found in the frameworks of both the heavy and lightchain v-domains of mVH/mVL were absent in all library-derived anddesigner leads (FIG. 17). GE epitope content was also found to besignificantly increased (from 3 to ≥14 in all leads), particularly inthe VH regions of lead clones where GE content increased from 0 to 9 inall leads, and TCED+ epitopes were reduced from in all leads from 3 to 2(Table 8). Importantly, however, multiple foreign epitopes were alsoeliminated by germlining mutations found in the CDRs of lead clones. Forexample, a TCED+ peptide ‘LVHSNGNTY’ (SEQ ID NO: 116) found in theLCDR-1 of mVH/mVL (and, therefore, in any forms of VxP037 previouslyhumanized by CDR grafting) was eliminated in the majority of lead clonesby the mutation V>L at position 2 and N>Y at position 7 (Tables 4 and5). Similarly, the LCDR2 from the mVH/mVL sequence encoded for threeforeign epitope peptides spanning the VL Framework 2-LCDR2-Framework 3.These included two HAF peptides (‘LLIYKVSYR’ (SEQ ID NO: 117) and‘YRFSGVPDR’ (SEQ ID NO: 118)) and one LAF peptide (‘LIYKVSYRF’ (SEQ IDNO: 119)). Insertion of the LCDR2 into the human germline frameworkIGKV2-28 did not fully remove this issue, as the total sequence‘LLIYKVSYRFSGVPDR’ (SEQ ID NO: 120) from mVH/mVL maintains 100% identityin the IGKV2-28 grafted sequence (Table 2). One HAF and two LAF peptideswere still found in this region for clones A-D5, A-D5.4 and A-D5.16,while the HAF sequence ‘YRFSGVPDR’ (SEQ ID NO: 118) was deleted by themutation Y>N at position 1. Surprisingly however, the LCDR2 sequenceKVSNRFS (SEQ ID NO: 85) that had been identified in the functionalbinding population during library screening (Table 3), and contained thesingle human germlining mutation Y>N at position 4, was found to fullyameliorate all predicted foreign epitopes in this region (no HAF, LAF orTCED+ peptides predicted), while also generating two further GEsequences (FIG. 18). It was also observed that the lead clones A-D5 andall of its designer derivatives (Tables 4 and 5) retained an HAF peptide‘VGVYYCFQN’ (SEQ ID NO: 121) that was also TCED+, spanning the VLFramework 3 and LCDR3 regions. The mutation of V at position 1 of thispeptide to A, T or F were all found to disrupt predicted epitopestructure and remove the immunogenicity risk of this peptide.

The findings above allowed the design of a maximally deimmunised lightchain sequence which was paired with the A-D5.16 VH sequence (Table 4)to form the clone ‘A-D5.16-DI’. A-D5.16-DI contained the LCDR sequence‘RSSQSLLHSAGYNYLH’ (SEQ ID NO: 82), the LCDR2 sequence ‘KVSNRFS’ (SEQ IDNO: 85) and the Framework 2-LCDR3 sequence ‘AGVYYCFQNTHTPRT’ (SEQ ID NO:122) (Framework 2 residues underlined). This clone was readily expressedin IgG1null format and was found to retain target binding affinityagainst human, cyno and mouse CD47 (FIG. 18. A-C) comparable to themVH/mVL IgG (reduced on mouse FIG. 18C). A-D5.16-DI was also found toexhibit improved CD47-SIRPα blockade in comparison to A-D5.16 andcomparable blockade to mVH/mVL against human and cyno CD47, slightlyreduced on mouse (FIG. 19). These findings thereby led to a deimmunizedclone A-D5.4-DI with a light chain design containing the LCDR sequence“RSSQSLLHSNGYTYLH” (SEQ ID NO: 52) (or SSQSLLHSNGYTY [SEQ ID NO: 92])using the AHo definition), the LCDR2 sequence “KVSNRFS” (SEQ ID NO: 85)and the Framework 2-LCDR3 sequence “AGVYYCFQNTHTPRT” (SEQ ID NO: 122)(Framework 2 residues underlined).

Phagocytosis of Cancer Cells

Studies were performed to examine the relative potency of CD47 blockadein driving the phagocytosis of HL60 human cancer cells by human primarymacrophages. As shown in FIG. 20A, in IgG4 (S228P) format—mVH/mVL, A-D5,A-D5.4 and A-D5.16 all drove significant phagocytosis at allconcentrations tested. A-D5 in IgG1null format did not show anysignificant potency at 20 μg/ml, demonstrating the necessity of Fc gammareceptor 1 binding affinity to provoke phagocytosis. Unexpectedly, IgG4A-D5, A-D5.4 and A-D5.16 all drove significantly more potentphagocytosis at both 1 and 10 μg/ml, when compared to mVH/mVL. Thisphenomenon was then examined for IgG4 A-D5 and mVH/mVL across 4 separatedonors of human macrophage (FIG. 20B). This analysis showed that thehigher potency shown in FIG. 20A was correct, with A-D5 beingsignificantly more potent in all donors (FIG. 20B).

Although the present invention has been described with reference topreferred or exemplary embodiments, those skilled in the art willrecognize that various modifications and variations to the same can beaccomplished without departing from the spirit and scope of the presentinvention and that such modifications are clearly contemplated herein.No limitation with respect to the specific embodiments disclosed hereinand set forth in the appended claims is intended nor should any beinferred.

All documents cited herein are incorporated by reference in theirentirety.

TABLE 1Amino acid sequences murine anti-CD47 CDRs as defined here (“Unified” scheme)in comparison to alternative definitions. SEQ ID NOs are shown in brackets.Scheme HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Unified GYTFTNYYVFIGDINPVNGDTNFNEKFKN GGYTMDY RSSQSLVHSNGNTYLH KVSYRFS SQNTHVPRT (4) (123)(5) (9) (10) (11) Rabat NYYVF DINPVNGDTNFNEKFKN GGYTMDY RSSQSLVHSNGNTYLHKVSYRFS SQNTHVPRT (124) (125) (5) (9) (10) (11) Chotia GYTFTNY NPVNGDGGYTMDY RSSQSLVHSNGNTYLH KVSYRFS SQNTHVPRT (126) (127) (5) (9) (10) (11)IMGT GYTFTNYY INPVNGDT TRGGYTMDY QSLVHSNGNTY KVS SQNTHVPRT (128) (129)(130) (131) (11) AHo GSGYTFTNYY INPVNGDTNFNEKFKN GGYTMD SSQSLVHSNGNTYKVSYRFS NTHVPR (15) (132) (16) (19) (10) (20) AbM GYTFTNYYVF DINPVNGDTNGGYTMDY RSSQSLVHSNGNTYLH KVSYRFS SQNTHVPRT (4) (133) (5) (9) (10) (11)Contact TNYYVF IGDINPVNGDTN TRGGYTMD HSNGNTYLHWY LLIYKVSYRF SQNTHVPR(134) (135) (136) (137) (138) (139)

TABLE 2Amino acid sequence of VXP037 murine anti-CD47 v-domains (mVH/mVL)and human germline CDR grafts (VH1/VL1). SEQ ID NOs are shown in brackets.V-DOMAIN Human germline¹ Amino acid sequence² CD47-mVH n/aEVQLQQFGAELVKPGASMKLSCKAS GYTFTNYYVF WVKQRPGQGLEW IGDINPVNGDTNFNEKFKNKATLTVDKSSTTTYLQLSSLTSEDSAVYYCTR GGYTMDY WGQGTLVTVSS (140) CD47-VH1IGHV5-51 EVQLVQSGAEVKKPGESLKISCKGS GYTFTNYYVF WVRQMPGKGLEWIGDINPVNGDTNFNESFQG QVTISADKSISTAYLQWSSLKASDTAMYYCAR GGYTMDY WGQGTLVTVSS(141) CD47-mVL n/a DVVMTQTPLSLSVSLGDQASISC RSSQSLVHSNGNTYLHWYLQKPGQSPKLLIY KVSYRFS GVPDR FSGSGSGTDFTLKISRVEAEDLGVYFC SQNTHVPRTFGQGTKVEIK (142) CD47-VL1 IGKV2-28 DIVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLH WYLQKPGQSPQLLIY KVSYRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQNTHVPRT FGQGTKVIEK (143) ₁Human germlinedefinitions used for grafting, based on IMGT system. ²CDR residues arein bold and underlined. As noted above, the “Unified” CDR definitionsused in this manuscript are an expanded definition in comparison to theclassical Kabat definition.

TABLE 3 Amino acid sequences of unique CDRs (using Unified definition)from 854 unique anti-CD47 v-domains. SEQ ID NOs are shown in brackets.LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 RSSHSLVHSNGNTYLH KGSNRAS FQNTHTPRTGYNFTNYYIF MGDINPFNGDTNFNPSFQG GGFTMDY (144) (67) (54) (145) (146) (66)RSSQSFLHSNGNNYLH KGSNRFS LQNTHTPRT GYRFTNYYIF MGDINPGDGDTNFNPSFQGGGHTMDY (147) (47) (148) (149) (150) (151) RSSQSLLHSNGNNYLD KGSNRSSMQALHTPWT GYRFTNYYVF MGDINPGNGDTNFNPSFQG GGITMDY (152) (153) (154) (155)(156) (157) RSSQSLLHSNGNNYLH KGSYRAS MQALHVPRT GYSFNNYYIFMGDINPGNGDTNYNPSFQG GGQTMDQ (158) (58) (159) (160) (161) (162)RSSQSLLHSNGNTYLD KGSYRFS MQALQVPWT GYSFTNYYIF MGDINPGNGDTNYSPSFQGGGYIMDY (163) (63) (164) (43) (165) (166) RSSQSLLHSNGNTYLH KGSYRLSMQATHVPWT GYSFTSYYIF MGDINPGNGDTRFNPSFQG GGYTADY (167) (168) (169) (170)(171) (172) RSSQSLLHSNGSNYLH KVSNRAS MQATQVPWT GYSFTSYYIVMGDINPGNGDTRFSPSFQG GGYTLDY (173) (174) (175) (176) (177) (178)RSSQSLLHSNGYNYLH KVSNRFS MQNLQTPRT GYSFTSYYVF MGDINPGNGDTRYNPSFQGGGYTMDA (46) (85) (179) (180) (181) (182) RSSQSLLHSNGYTYLD KVSNRLSMQNTHIPRT GYSFTSYYVG MGDINPGNGDTRYSPSFQG GGYTMDE (183) (53) (184) (185)(186) (187) RSSQSLLHSNGYTYLH KVSYRAS MQNTHTLRT GYTFTNYYIFMGDINPGNSDTNFNPSFQG GGYTMDF (52) (188) (189) (49) (190) (191)RSSQSLVHSNGNNYLD KVSYRLS MQNTHTPRT GYTFTSYYIF MGDINPGNSDTNYNPSFQGGGYTMDH (192) (193) (194) (195) (196) (197) RSSQSLVHSNGNNYLH LGSNRASMQNTHVPRT GYTFTSYYVF MGDINPVDGDTKYNPSFQG GGYTMDI (201) (72) (198) (199)(200) (70) RSSQSLVHSNGNTYLD LGSNRFS MQNTQTPRT MGDINPVDGDTNFNPSFQGGGYTMDK (62) (71) (202) (203) (56) RSSQSLVHSNGYNYLH LGSNRLS MQTTHTPRTMGDINPVDGDTNFSPSFQG GGYTMDL (207) (77) (204) (205) (206)RSSQSLVHSNGYTYLD LGSYRAS MQTTQIPRT MGDINPVDGDTNYNPSFQG GGYTMDM (212)(208) (209) (210) (211) RSSQSLVHSNGYTYLH LGSYRFS SQATHFPRTMGDINPVDGDTNYSPSFQG GGYTMDN (57) (213) (214) (215) (216) LGSYRLSSQATQTPRT MGDINPVDGDTRFNPSFQG GGYTMDQ (217) (73) (218) (219) LVSNRASSQNIQTPRT MGDINPVDGDTRYNPSFQG GGYTMDR (220) (221) (222) (51) LVSNRFSSQNLHTPRT MGDINPVDGDTRYSPSFQG GGYTMDT (223) (224) (225) (226) LVSNRLSSQNLQTPRT MGDINPVDSDTKFNPSFQG GGYTMDV (227) (228) (229) (230) LVSYRASSQNMHTPRT MGDINPVDSDTNFNPSFQG GGYTMDW (231) (232) (233) (234) MGSNRFSSQNTHFPRT MGDINPVDSDTNYNPSFQG GGYTMGK (235) (236) (237) (61) MGSYRLSSQNTHVPWT MGDINPVDSDTNYSPSFQG GGYTPDY (238) (239) (240) (45) MVSNRFSSQNTQAPRT MGDINPVDSDTRFNPSFQG GGYTRDY (241) (242) (243) (244) SQNTQTPRTMGDINPVDSDTRYNPSFQG GGYTTDS (59) (245) (246) SQNTQTPWTMGDINPVDSDTRYSPSFQG GGYTTDW (247) (248) (249) SQNTQVPRTMGDINPVNGDTKYNPSFQG GGYTTDY (250) (251) (252) SQSTHVPRTMGDINPVNGDTNFSPSFQG GGYVMDY (253) (254) (255) SQTTHIPRTMGDINPVNGDTNYNPSFQG (256) (257) SQTTHVPRT MGDINPVNGDTNYSPSFQG (258) (44)SQTTQTPRT MGDINPVNGDTRFNPSFQG (259) (260) TQNTHTPRT MGDINPVNGDTRFSPSFQG(261) (262) VQNTQVPRT MGDINPVNGDTRYNPSFQG (263) (264)MGDINPVNGDTRYSPSFQG (265) MGDINPVNSDTKYNPSFQG (266) MGDINPVNSDTNFNPSFQG(267) MGDINPVNSDTNYSPSFQG (268) MGDINPVNSDTRFNPSFQG (269)MGDINPVNSDTRFSPSFQG (270) MGDINPVNSDTRYNPSFQG (271) MGDINPVNSDTRYSPSFQG(272) MGDIYPGNSDTKYNPSFQG (273) MGDIYPVNGDTRYNPSFQG (274)MGIINPGNGDTRYNPSFQG (275) MGIINPVDGDTKYNPSFQG (276) MGIINPVDGDTNYSPSFQG(277) MGIINPVDGDTRFNPSFQG (278) MGIINPVDGDTRFSPSFQG (279)MGIINPVDGDTRYNPSFQG (74) MGIINPVDGDTRYSPSFQG (65) MGIINPVDSDTNYNPSFQG(280) MGIINPVDSDTRFNPSFQG (281) MGIINPVDSDTRYNPSFQG (282)MGIINPVDSDTRYSPSFQG (283) MGIINPVNGDTKFNPSFQG (284) MGIINPVNGDTKYNPSFQG(285) MGIINPVNGDTKYSPSFQG (286) MGIINPVNGDTNFNPSFQG (287)MGIINPVNGDTNFSPSFQG (288) MGIINPVNGDTNYNPSFQG (60) MGIINPVNGDTNYSPSFQG(289) MGIINPVNGDTRFNPSFQG (290) MGIINPVNGDTRFSPSFQG (291)MGIINPVNGDTRYNPSFQG (292) MGIINPVNGDTRYSPSFQG (293) MGIINPVNSDTKYNPSFQG(294) MGIINPVNSDTNFNPSFQG (295) MGIINPVNSDTNYSPSFQG (296)MGIINPVNSDTRFSPSFQG (297) MGIINPVNSDTRYNPSFQG (298) MGIINPVNSDTRYSPSFQG(299) MGNINPVDGDTRYNPSFQG (300) MGVINPVNSDTNYNPSFQG (301)

TABLE 4Amino acid sequences of CDRs (using Unified definition) of unique,library-derived and designer, CD47-SIRPα interaction-blocking anti-CD47IgGs. SEQ ID NOs are shown in brackets. Clone name LCDR1 LCDR2 LCDR3HCDR1 HCDR2 HCDR3 D-H3 RSSQSLLHSNGYNYLH KGSNRFS SQNLHVPRT GYSFTNYYIFMGDINPVNGDTNYSPSFQG GGYTPDY (46) (47) (48) (43) (44) (45) A-D5RSSQSLLHSNGYTYLH KVSNRLS FQNTHTPRT GYTFTNYYIF MGIINPVDGDTNYNPSFQGGGYTMDR (52) (53) (54) (49) (50) (51) G-B6 RSSQSLVHSNGYTYLH KGSYRASSQNTQTPRT GYSFTNYYIF IGDINPVNGDTNFSPSFQG GGYTMDK (57) (58) (59) (43)(55) (56) F-E7 RSSQSLVHSNGNTYLD KGSYRFS SQATHTPRT GYSFTNYYIFMGIINPVNGDTNYNPSFQG GGYTMGK (62) (63) (64) (43) (60) (61) VH-A1/RSSQSLLHSNGYNYLH KGSNRAS SQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYSPSFQGGGFTMDY VL-B1 (46) (67) (68) (43) (65) (66) MH RSSQSLLHSNGYNYLH LGSNRFSSQNTQTPRT GYSFTNYYIF IGIINPVDGDTRYSPSFQG GGYTMDI (46) (71) (59) (43)(69) (70) TTP RSSQSLLHSNGYNYLH LGSNRAS SQATQTPRT GYSFTNYYIFMGIINPVDGDTRYSPSFQG GGYTMDI (46) (72) (73) (43) (65) (70) A-D5.1RSSQSLLHSNGYTYLH KVSNRLS FQNTHTPRT GYSFTNYYIF MGIINPVDGDTNYNPSFQGGGYTMDR (52) (53) (54) (43) (50) (51) A-D5.2 RSSQSLLHSNGYTYLH KVSNRLSFQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYNPSFQG GGYTMDR (52) (53) (54) (43)(74) (51) A-D5.3 RSSQSLLHSNGYTYLH KVSNRLS FQNTHTPRT GYSFTNYYIFMGIINPVDGDTRYSPSFQG GGYTMDR (52) (53) (54) (43) (65) (51) A-D5.4RSSQSLLHSNGYNYLH KVSNRLS FQNTHTPRT GYTFTNYYIF MGIINPVDGDTNYNPSFQGGGYTMDR (46) (53) (54) (49) (50) (51) A-D5.5 RSSQSLLHSNGYNYLH KGSNRLSFQNTHTPRT GYTFTNYYIF MGIINPVDGDTNYNPSFQG GGYTMDR (46) (75) (54) (49)(50) (51) A-D5.6 RSSQSLLHSNGYNYLH KGSNRLS FQNTQTPRT GYTFTNYYIFMGIINPVDGDTNYNPSFQG GGYTMDR (46) (75) (76) (49) (50) (51) A-D5.7RSSQSLLHSNGYNYLH LGSNRLS FQNTQTPRT GYTFTNYYIF MGIINPVDGDTNYNPSFQGGGYTMDR (46) (77) (76) (49) (50) (51) A-D5.8 RSSQSLLHSQGYTYLH KVSNRLSFQNTHTPRT GYTFTNYYIF MGIINPVDGDTNYNPSFQG GGYTMDR (78) (53) (54) (49)(50) (51) A-D5.9 RSSQSLLHSNGYTYLH KVSNRLS FQQTHTPRT GYTFTNYYIFMGIINPVDGDTNYNPSFQG GGYTMDR (52) (53) (79) (49) (50) (51) A-D5.10RSSQSLLHSQGYTYLH KVSNRLS FQQTHTPRT GYTFTNYYIF MGIINPVDGDTNYNPSFQGGGYTMDR (78) (53) (79) (49) (50) (51)

TABLE 5Amino acid sequences of CDRs (using Unified definition) of unique,A-D5-derived, CD47-SIRPα interaction-blocking, designer anti-CD47 IgGs.SEQ ID NOs are shown in brackets. Clone name LCDR1 LCDR2 LCDR3 HCDR1HCDR2 HCDR3 A-D5.11 RSSQSLLHSNGYNYLH KVSNRLS FQNTHTPRT GYSFTNYYIFMGIINPVDGDTNYNPSFQG GGYTMDR (46) (53) (54) (43) (50) (51) A-D5.12RSSQSLLHSNGYNYLH KVSNRLS FQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYNPSFQGGGYTMDR (46) (53) (54) (43) (74) (51) A-D5.13 RSSQSLLHSNGYNYLH KVSNRLSFQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYSPSFQG GGYTMDR (46) (53) (54) (43)(65) (51) A-D5.14 RSSQSLLHSSGYNYLH KVSNRLS FQNTHTPRT GYSFTNYYIFMGIINPVDGDTRYSPSFQG GGYTMDR (80) (53) (54) (43) (65) (51) A-D5.15RSSQSLLHSGGYNYLH KVSNRLS FQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYSPSFQGGGYTMDR (81) (53) (54) (43) (65) (51) A-D5.16 RSSQSLLHSAGYNYLH KVSNRLSFQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYSPSFQG GGYTMDR (82) (53) (54) (43)(65) (51) A-D5.17 RSSQSLLHSTGYNYLH KVSNRLS FQNTHTPRT GYSFTNYYIFMGIINPVDGDTRYSPSFQG GGYTMDR (83) (53) (54) (43) (65) (51) A-D5.18RSSQSLLHSNAYNYLH KVSNRLS FQNTHTPRT GYSFTNYYIF MGIINPVDGDTRYSPSFQGGGYTMDR (84) (53) (54) (43) (65) (51)

The invention claimed is:
 1. A method for treating cancer in a subject,comprising administering to the subject an effective amount of anantibody molecule that specifically binds to human Integrin AssociatedProtein (CD47) and cynomolgus monkey CD47, or an antigen-binding portionthereof, wherein the antibody molecule or antigen-binding portioncomprises: (a) a heavy chain variable region comprising a heavy chaincomplementarity determining region (HCDR) 1 of SEQ ID NO: 49, a HCDR2 ofSEQ ID NO: 50, and a HDCR3 of SEQ ID NO: 51; and a light chain variableregion comprising a light chain complementarity determining region(LCDR) 1 of SEQ ID NO: 52, a LCDR2 of SEQ ID NO: 53, and a LDCR3 of SEQID NO: 54; or (b) a heavy chain variable region comprising a HCDR1 ofSEQ ID NO: 49, a HCDR2 of SEQ ID NO: 50, and a HDCR3 of SEQ ID NO: 51;and a light chain variable region comprising a LCDR1 of SEQ ID NO: 52, aLCDR2 of SEQ ID NO: 85, and a LDCR3 of SEQ ID NO:
 54. 2. The method ofclaim 1, wherein the cancer is pancreatic cancer, melanoma, breastcancer, lung cancer, bronchial cancer, colorectal cancer, prostatecancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain orcentral nervous system cancer, peripheral nervous system cancer,esophageal cancer, cervical cancer, uterine or endometrial cancer,cancer of the oral cavity or pharynx, liver cancer, kidney cancer,testicular cancer, biliary tract cancer, small bowel or appendix cancer,salivary gland cancer, thyroid gland cancer, adrenal gland cancer,osteosarcoma, chondrosarcoma, or cancer of hematological tissues.
 3. Themethod of claim 1, wherein the antibody molecule or antigen-bindingportion is humanized or chimeric.
 4. The method of claim 1, wherein theheavy chain variable region, the light chain variable region, or boththe heavy chain variable region and the light chain variable region ofthe antibody molecule or antigen-binding portion comprise a humanvariable domain framework scaffold into which the CDRs have beeninserted.
 5. The method of claim 1, wherein the heavy chain variableregion of the antibody molecule or antigen-binding portion comprises anIGHV5-51 human germline scaffold into which the HCDR1, HCDR2 and HCDR3sequences have been inserted.
 6. The method of claim 1, wherein thelight chain variable region of the antibody molecule or antigen-bindingportion comprises an IGKV2-28 human germline scaffold into which theLCDR1, LCDR2 and LCDR3 sequences have been inserted.
 7. The method ofclaim 1, wherein the antibody molecule or antigen-binding portioncomprises an immunologically inert constant region.
 8. The method ofclaim 1, wherein the antibody molecule or antigen-binding portion is aFab fragment, a F(ab)₂ fragment, an Fv fragment, a tetrameric antibody,a tetravalent antibody, a multispecific antibody or an scFv.
 9. Themethod of claim 8, wherein the multispecific antibody is a bivalentantibody.
 10. The method of claim 1, wherein the antibody molecule orantigen-binding portion specifically binds to mouse CD47.
 11. A methodfor treating cancer in a subject, comprising administering to thesubject an effective amount of a pharmaceutical composition comprising(i) an antibody molecule that specifically binds to human IntegrinAssociated Protein (CD47) and cynomolgus monkey CD47, or anantigen-binding portion thereof, wherein the antibody molecule orantigen-binding portion comprises: (a) a heavy chain variable regioncomprising a heavy chain complementarity determining region (HCDR) 1 ofSEQ ID NO: 49, a HCDR2 of SEQ ID NO: 50, and a HDCR3 of SEQ ID NO: 51;and a light chain variable region comprising a light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 52, a LCDR2 ofSEQ ID NO: 53, and a LDCR3 of SEQ ID NO: 54; or (b) a heavy chainvariable region comprising a HCDR1 of SEQ ID NO: 49, a HCDR2 of SEQ IDNO: 50, and a HDCR3 of SEQ ID NO: 51; and a light chain variable regioncomprising a LCDR1 of SEQ ID NO: 52, a LCDR2 of SEQ ID NO: 85, and aLDCR3 of SEQ ID NO: 54; and (ii) a pharmaceutically acceptableexcipient.
 12. The method of claim 11, wherein the cancer is pancreaticcancer, melanoma, breast cancer, lung cancer, bronchial cancer,colorectal cancer, prostate cancer, stomach cancer, ovarian cancer,urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer ofhematological tissues.
 13. The method of claim 11, wherein the antibodymolecule or antigen-binding portion is humanized or chimeric.
 14. Themethod of claim 11, wherein the heavy chain variable region, the lightchain variable region, or both the heavy chain variable region and thelight chain variable region of the antibody molecule or antigen-bindingportion comprise a human variable domain framework scaffold into whichthe CDRs have been inserted.
 15. The method of claim 11, wherein theheavy chain variable region of the antibody molecule or antigen-bindingportion comprises an IGHV5-51 human germline scaffold into which theHCDR1, HCDR2 and HCDR3 sequences have been inserted.
 16. The method ofclaim 11, wherein the light chain variable region of the antibodymolecule or antigen-binding portion comprises an IGKV2-28 human germlinescaffold into which the LCDR1, LCDR2 and LCDR3 sequences have beeninserted.
 17. The method of claim 11, wherein the antibody molecule orantigen-binding portion comprises an immunologically inert constantregion.
 18. The method of claim 11, wherein the antibody molecule orantigen-binding portion is a Fab fragment, a F(ab)₂ fragment, an Fvfragment, a tetrameric antibody, a tetravalent antibody, a multispecificantibody or an scFv.
 19. The method of claim 18, wherein themultispecific antibody is a bivalent antibody.
 20. The method of claim11, wherein the antibody molecule or antigen-binding portionspecifically binds to mouse CD47.